Limitation on the use of the horizontal clinostat as a gravity compensator

If the horizontal clinostat effectively compensates for the influence of the gravity vector on the rotating plant, it should make the plant unresponsive to whatever chronic acceleration may be applied transverse to the axis of clinostat rotation. This was tested by centrifuging plants while they were growing on clinostats. For a number of morphological end-points of development the results depended on the magnitude of the applied g-force. Therefore, gravity compensation by the clinostat was incomplete. This conclusion is in agreement with results of satellite experiments which are reviewed.     

Effects of clinorotation on the structure of xylem in Arabidopsis roots]

Lignin, which is the most important component of vascular tissue, is essential material for the structural support of higher plants and considered to play a critical role in evolution of land plants. It has been postulated that development of secondary wall is mediated by gravity. However, effects of gravity on the development and the morphology of secondary cell wall have not been well investigated. In this study, effects of averaging of gravity vector using 2-D clinostat rotation on the structure of xylem was examined. The results indicated that the morphology of xylem is regulated by the orientation of gravity vector.     

Effects of 3-D clino-rotation on gene expression in human fibroblast cells

Continuous variation in the direction of the gravity vector leads to various cellular responses including modulation of gene expression. Complementary DNA (cDNA) array analyses are available to observe the variation of gene expression under different conditions. In this study, expression levels of 588 representative genes were compared using the Atlas human cDNA expression array in human fibroblast cells with and without 3-dimensional (3-D) clinostat. Five upregulated and 8 downregulated genes were detected. Among these genes, upregulation of XRCC1, and downregulation of ERB-B2 and p21(Cip1/Waf1) were confirmed by RT-PCR. These results suggested that the gene expression levels of XRCC1, ERB-B2 and p21(Cip1/Waf1) were modulated by vector-averaged microgravity induced by 3-D clinostat in human fibroblast cells. Our findings may be a basis for the biological study of 3-D culture systems.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Gravity can induce epinastic bending of isolated leaves

Abstract Isolated leaves of Plectranthus fruticosus were grown in cubic plastic cuvettes, and were supplied via their cut petioles with nutrient solution and indole-3-acetic acid (10^?6m ). Holes bored in the cuvette walls allowed the petioles to be oriented at approximately 60°, 90° or 120° to the vertical. Growth of the leaves initially oriented at angles of 60° and 90°, which simulated the situation in the intact plant, did not result in epinastic bending of the petiole. Inversion of the leaves (adaxial surface of the petiole downwards) and orientation of the adaxial/abaxial surfaces of the horizontal petiole parallel to the gravity vector, however, yielded strong epinastic bending of the petioles. In the latter case, this bending was not in the direction of the gravity vector (evidence for point (iii), below). Furthermore, epinastic bending occurred, when the isolated leaves were rotated on a clinostat (petioles parallel to the rotation axis or inclined to the rotation axis at an angle of 30°; 3 r.p.m.). Since a possible influence of the shoot was excluded, it is concluded that (i) perception and response are restricted to the leaf, (ii) gravity alone is sufficient to induce epinasty, (iii) a gravitropic component of the response can probably be excluded. The clinostat induced epinasty may not have been caused by nullifying the effect of gravity but due to continuous gravistimulation of the leaf.     

轮藻假根中的平衡石在回转器水平回转时的运动(英文)

利用回转器重现了在TEXUS火箭抛物线飞行的微重力实验中轮藻假根内平衡石向假根基部方向的运动。在快速回转器上回转15 min时,假根中的平衡石复合体中心离假根顶端的距离比在原来沿重力方向生长的假根中的距离增加了60%。细胞松弛素D的实验证实平衡石的这种运动是和肌动蛋白丝相关,而且在重力场中作用于平衡石的向基力也是肌动蛋白丝产生的。因此回转器和细胞松弛素D的实验证实了在地球上,平衡石的位置取决于作用方向相反的重力和肌动蛋白丝作用力的动态平衡的假说。然后在快速回转器上,平衡石中心在一个新的位置上维持了30 min左右的稳定,也就是出现了一个新的动态平衡状态。这一新的状态是在原先的向着假根顶端的重力和向着假根基部的肌动蛋白丝作用力的平衡在回转器上被打破后再经约有15 min时达到的。更进一步的快速回转器实验还展示了可能因平衡石位置的这一变化而启动的肌动蛋白丝的再组织和由此产生的平衡石向假根顶端方向再转运的过程。快速和慢速回转器实验在这里的结果有差异,推测是和回转器上颗粒的振幅随回转器转速的增加而减小有关。加之,轮藻假根的单细胞性质,因此在假根处于回转轴上时,快速回转器是更适合这项模拟失重的研究。总之,在失重条件下平衡石和肌动蛋白丝的关系是可以利用回转器来研究的。     

轮藻假根中的平衡石在回转器水平回转时的运动

利用回转器重现了在ＴＥＸＵＳ火箭抛物线飞行的微重力实验中轮藻假根内平衡石和假根基部方向的运动。在快速回转器上回转１５ｍｉｎ时,假根中的平衡石复合体中心离假根顶端的距离比在原来沿重力方向生长的假根中的距离增加了６０％。细胞松弛素Ｄ的实验证实平衡石的这种运动是和肌动蛋白丝相关,而且在重力场中作用于平衡石的向基力也是肌动蛋白丝产生的。因此回转器和细胞松弛素Ｄ的实验证实了在地球上,平衡石的位置取决于作用方     

Automorphosis of etiolated pea seedlings in space is simulated by a three-dimensional clinostat and the application of inhibitors of auxin polar transport

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under microgravity conditions in space show automorphosis: bending of epicotyls, inhibition of hook formation and changes in root growth direction. In order to determine the mechanisms of microgravity conditions that induce automorphosis, we used a three-dimensional clinostat and obtained the successful induction of automorphosis-like growth of etiolated pea seedlings. Kinetic studies revealed that epicotyls bent at their basal region towards the clockwise direction far from the cotyledons from the vertical line (0 degrees) at approximately 40 degrees in seedlings grown both at 1 g and in the clinostat within 48 h after watering. Thereafter, epicotyls retained this orientation during growth in the clinostat, whereas those at 1 g changed their growth direction against the gravity vector and exhibited a negative gravitropic response. On the other hand, the plumular hook that had already formed in the embryo axis tended to open continuously by growth at the inner basal portion of the elbow; thus, the plumular hook angle initially increased; this was followed by equal growth on the convex and concave sides at 1 g, resulting in normal hook formation; in contrast, hook formation was inhibited on the clinostat. The automorphosis-like growth and development of etiolated pea seedlings was induced by auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid), but not by anti-auxin (p-chlorophenoxyisobutyric acid) at 1 g. An ethylene biosynthesis inhibitor, 1-aminooxyacetic acid, inhibited hook formation at 1 g, and ethylene production of etiolated seedlings was suppressed on the clinostat. Clinorotation on the clinostat strongly reduced the activity of auxin polar transport of epicotyls in etiolated pea seedlings, similar to that observed in space experiments (Ueda J, Miyamoto K, Yuda T, Hoshino T, Fujii S, Mukai C, Kamigaichi S, Aizawa S, Yoshizaki I, Shimazu T, Fukui K (1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J Plant Res 112: 487492). These results suggest that clinorotation on a three-dimensional clinostat is a valuable tool for simulating microgravity conditions, and that automorphosis of etiolated pea seedlings is induced by the inhibition of auxin polar transport and ethylene biosynthesis.     

Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells

Clinostat is an effective, ground-based tool which can be used to verify data from space flight, and to test hypotheses and experimental conditions for eventual space flights. Rotation in clinostat appears to mimic the microgravity environment by nulling the gravitational vector by continuous averaging. In the present study, we exposed osteoblast-like ROS 17/2.8 cells to a vector-averaged gravity environment in a clinostat and found that the cells undergo apoptotic death during the first 24 hr of clino-rotation. We suggest that apoptosis might be one of the mechanisms for reduced bone formation as observed in actual space flights.     

Vector-averaged gravity does not alter acetylcholine receptor single channel properties.

To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.     

Enhancement of phototropic response to a range of light doses in Triticum aestivum coleoptiles in clinostat-simulated microgravity

The phototropic dose-response relationship has been determined for Triticum aestivum cv. Broom coleoptiles growing on a purpose-built clinostat apparatus providing gravity compensation by rotation about a horizontal axis at 2 rev·min^-1. These data are compared with data sets obtained with the clinostat axis vertical and stationary, as a 1·g control, and rotating vertically to examine clinostat effects other than gravity compensation. Triticum at 1·g follows the wellestablished pattern of other cereal coleoptiles with a first positive curvature at low doses, followed by an indifferent response region, and a second positive response at progressively increasing doses. However, these response regions lie at higher dose levels than reported for Avena. There is no significant difference between the responses observed with the clinostat axis vertical in the rotating and stationary modes, but gravity compensation by horizontal rotation increases the magnitude of first and second positive curvatures some threefold at 100 min after stimulation. The indifferent response is replaced by a significant curvature towards the light source, but remains apparent as a reduced curvature response at these dose levels.     

Experiments with osteoblasts cultured under varying orientations with respect to the gravity vector

Substrate attachment is crucial for normal growth and differentiation of many cell types. To better understand the role of gravity in osteoblast attachment and growth in vitro, 17-day-old embryonic chick calvarial osteoblasts were subjected to directional variations with respect to gravity. Osteoblasts, grown in MEM or DME supplemented with 10% FBS and attached to type I collagen-coated coverslips, were loaded into cylindrical containers completely filled with medium and oriented so that cells were either atop or beneath, or coverslips continuously rotated (∼2 rpm) in a clinostat, thereby continuously changing their orientation with respect to gravity. Cells in these three conditions were collected daily for up to 6 days, and cell viability, two osteoblast functions, and proliferation were assessed. Data suggest the number and function of attached osteoblasts is unaltered by inversion or clino-rotation in initially confluent cultures. In sparsely plated cultures, however, osteoblast viability was significantly decreased (∼50%) in inverted and rotated cultures during the first 3 days of sampling, but from days 4–6 no significant difference was found in viable cell number for the three conditions. Decreases in viable cell number within the first days of the experiments could result from death followed by detachment, detachment followed by death, differences in proliferation rate, or lag-phase duration. To help distinguish among these, BrdU labeling for 2 or 24 hr was used to assess cell proliferation rate. Log-phase growth rates were calculated and were unchanged among the three conditions tested. These results point to an increase in lag-phase duration in inverted and rotated cultures. In summary, changing the cell-substrate attachment direction with respect to gravity causes an immediate response in the form of diminished viable osteoblast number in sparse, early cultures, but the effect disappears after 3–4 days and does not occur in mature, confluent cultures. This revised version was published online in September 2006 with corrections to the Cover Date.     

The dynamics of a discrete geotropic sensor subject to rotation-induced gravity compensation

A clinostat achieves gravity compensation by providing circular rotation with uniform speed, about a horizontal axis. The dynamics of an assumed, discrete and free-moving subcellular gravity receptor, subject to clinostat rotation, is analyzed. The results imply that there is an optimum rotation rate; higher speeds result in circular motions with diameters more comparable to thermal noise fluctuations, but with greater linear velocities due to increasing centrifugal forces. An optimizing function is proposed. The nucleolus and mitochondrion is chosen as a gravity receptor for illustrating the use of this theory. The characteristics of their clinostat-induced motions are incorporated with experimental results on Avena plant shoots in an illustrative example.     

重力钝感的水稻突变体在重力和微重力条件下淀粉体的观察分析（简报）

植物向重性的研究一直受到关注,主要的研究集中在双子叶模式生物拟南芥中,而对单子叶植物的研究却很少。植物对重力感受的方式存在多种解释,但目前大量证据表明淀粉体一平衡石理论较为合理,它认为淀粉体作为平衡石在植物向重性反应中发挥了重要的作用。经过100多年的研究,现已从生理学与     

重力钝感的水稻突变体在重力和微重力条件下淀粉体的观察分析

植物向重性的研究一直受到关注,主要的研究集中在双子叶模式生物拟南芥中,而对单子叶植物的研究却很少。植物对重力感受的方式存在多种解释,但目前大量证据表明淀粉体—平衡石理论较为合理,它认为淀粉体作为平衡石在植物向重性反应中发挥了重要的作用。经过100多年的研究,现已从生理学与遗传学的角度证实了含有淀粉体的根冠中柱细胞和茎的内皮层细胞是植物重力感受的部位,淀粉体作为重力感受器被越来越多的实验证据证明。地球上重力无处不在,要研究微重力对植物体极性生长的影响只能借助于能模拟失重环境的回转器。近年来,人们对植物向重性机制的了解主要来自缺失或缺少     

Plastid position in Arabidopsis columella cells is similar in microgravity and on a random-positioning machine

 In order to study gravity effects on plant structure and function, it may become necessary to remove the g-stimulus. On Earth, various instruments such as clinostats have been used by biologists in an attempt to neutralize the effects of gravity. In this study, the position of amyloplasts was assayed in columella cells in the roots of Arabidopsisthaliana (L.) Heynh. seedlings grown in the following conditions: on Earth, on a two-dimensional clinostat at 1 rpm, on a three-dimensional clinostat (also called a random-positioning machine, or an RPM), and in space (true microgravity). In addition, the effects of these gravity treatments on columella cell area and plastid area also were measured. In terms of the parameters measured, only amyloplast position was affected by the gravity treatments. Plastid position was not significantly different between spaceflight and RPM conditions but was significantly different between spaceflight and the classical two-dimensional clinostat treatments. Flanking columella cells showed a greater susceptibility to changes in gravity compared to the central columella cells. In addition, columella cells of seedlings that were grown on the RPM did not exhibit deleterious effects in terms of their ultrastructure as has been reported previously for seedlings grown on a two-dimensional clinostat. This study supports the hypothesis that the RPM provides a useful simulation of weightlessness. Received: 5 January 2000 / Accepted: 22 February 2000     

Orientation of Paramecium under the conditions of weightlessness

A cell culture of Paramecium with a precise negative gravitaxis was exposed to 4 x l0(-6) g during a parabolic flight of a sounding rocket for 6 min. Computer image analysis revealed that without gravity stimulus the individual swimming paths remained straight. In addition, three reactions could be distinguished. For about 30 s, paramecia maintained the swimming direction they had before onset of low gravity. During the next 20 s, an approximate reversal of the swimming direction occurred. This period was followed by the expected random swimming pattern. Similar behavior was observed under the condition of simulated weightlessness on a fast-rotating clinostat. Control experiments on the ground under hyper-gravity on a low-speed centrifuge microscope and on a vibration test facility proved that the observed effects were caused exclusively by the reduction of gravity.     

Effects of prolonged omnilateral gravistimulation on the ultrastructure of statocytes and on the graviresponse of roots

Statocytes of vertically growing roots of Lepidium sativum L. exhibit a strict polarity: The nucleus is positioned near the proximal periclinal cell wall, amyloplasts are sedimented on a complex of rough endoplasmic reticulum (ER) consisting of parallel cisternae near the distal periclinal cell wall.When 24 h old, vertically grown roots are rotated for an additional 20 h on a horizontal clinostat, this polarity is destroyed. Furthermore, the prolonged omnilateral stimulation leads to a damage of the statocytes, which in some cases ends in the self-destruction of the sensitive cells. The different components of the ultrastructural respones of the statocytes are: Displacement of the nucleus; changes in amount and distribution of the ER; loss of amyloplast starch; confluence of lipid droplets to large aggregates: a considerable increase of the lytic compartment. In addition, even anticlinal cell walls may be lysed up to small stumps. As all these effects are clearly restricted to the statocytes, only these cells are able to respond to the continuously changing direction of the gravity vector, thus perceiving gravity as such.After being exposed horizontally, the graviresponse of rotated roots is delayed as compared to the controls. About 20% of the rotated roots do not respond (curve) at all, but grow perpendicular in relation to the gravity vector. Perception of gravity is inevitably correlated with the polarity and the integrity of the statocytes.Abbreviation ER endoplasmic reticulum A preliminary report was presented at the Fall Meeting of the German Society for Cell Biology in Salzburg, Austria, September 1979 (Hensel and Sievers 1979)This paper represents part of a dissertation (D 5) of W. H.     

Interaction of gravi- and phototropic stimulation in the response of maize (Zea mays L.) coleoptiles

The influence of gravitropic stimulation upon blue-light-induced first positive phototropism for stimulations in the same (light source and center of gravity opposite to each other) and in opposing directions was investigated in maize cole-optiles by measuring fluence-response patterns. As a result of gravitropic counterstimulation, phototropic bending was transient with maximum curvature occurring 100 min after stimulation. On a horizontal clinostat, however, the seedlings curved for 20 h. Gravistimulation in the opposite direction acted additively upon blue-light curvature. Gravistimulation in the same direction as phototropic stimulation produced a complex behaviour deviating from simple additivity. This pattern can be explained by a gravitropically mediated sensitization of the phototropic reaction, an optimal dependence of differential growth on the sum of photo-and gravistimulation, and blue-light-induced inhibition of gravitropic curvature at high fluences. These findings indicate that several steps of photo-and gravitransduction are separate. Preirradiation with red light desensitized the system independently of applied gravity-treatment, indicating that the site of red-light interaction is common to both transduction chains.Abbreviations BL blue light - G⁺ stimulation by light and gravity in the same direction (i.e. light source and center of gravity opposite to each other) - G^- stimulation by light and gravity in opposing directions