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.     

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight.

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.     

Interpreting Plant Responses to Clinostating: I. MECHANICAL STRESSES AND ETHYLENE

The severe epinasty and other symptoms developed by clinostated leafy plants could be responses to gravity compensation and/or the mechanical stresses of leaf flopping. Epinasty in cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxyvinylglycine and Ag⁺), confirming the role of ethylene in clinostat epinasty. To test the possibility that clinostat mechanical stresses (leaf flopping) cause ethylene production and, thus, epinasty, vertical plants were stressed with constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approximated but with a minimum of gravity compensation, by turning plants so their stems were horizontal, rotating them quickly about the stem axis, and then returning them to the vertical, repeating the treatment every four minutes (clinostat rotation time). None of these mechanical stresses produced significant epinasties, but vigorous hand-shaking (120 seconds per day) generated minor epinasties, as did Ag⁺ applied daily (concentrations high enough to cause leaf browning). Plants gently inverted every 20 minutes developed epinasty at about the same rate and to about the same extent as clinostated plants, but plants inverted every 20 minutes and immediately returned to the upright position did not become epinastic. It is concluded that clinostat epinasty is probably caused by disturbances in the gravity perception mechanism, rather than by leaf flopping.     

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.     

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     

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.     

Choice of rotation rate for the horizontal clinostat

A series of nine rates of rotation of the clinostat were tested to determine optimal and acceptable conditions for simulating weightlessness in plants. Young seedlings of wheat (Triticum aestivum L.) developed roots and coleoptiles of equal lengths and with the same orientation angles over a range of rotation rates from 0.25 to 480 minutes per revolution. Rates from 0.25 to 3 minutes per revolution provided for maximal epinastic curvatures of leaves and branches of Coleus blumei Benth. except for a reduced mean curvature of branches at 0.25 minute per revolution, due probably to physical disturbances in their growth. Smaller epinastic curvatures developed in both leaves and branches rotated at 15 minutes per revolution or more slowly. Indoleacetic acid-2-¹⁴C was used for measurements of extractable radioactivity in determining the reason for smaller curvatures of Coleus branches and tomato (Lycopersicon esculentum Mill.) leaves at the rotation rate of 60 minutes per revolution than at 1 minute per revolution. The cause was determined to be movement of some auxin from the upper into the lower side of a plagiotropic leaf or branch under the transient influence of gravity during rotation on the slower clinostat. A rotation period of 1 to 3 minutes was found to be acceptable for most plants.     

The effect of simulated microgravity on seed germination and seedling anatomy of Phaseolus vulgaris L

Seed germination and root anatomy were investigated in seedlings of Phaseolus vulgaris L. developed on a slowly rotating bi-dimensional clinostat and in 1g. Germination time, percent germination, curvature and anatomy of developing root apexes were monitored on the clinostat and compared with the control. Interesting differences were found in germination and root features of the seeds developed on the clinostat compared with 1g ones: the main being germination time, root cap formation, the quantity and distribution of amyloplasts in statocytes. The use of a software to quantitatively analyse root cap anatomy allowed us to detect some differences otherwise unlikely to highlight. Our results showed that prolonged rotation on a bi-dimensional clinostat has an effect on some aspects of germination and on the statocytes that continuously perceives gravity from ever-changing directions.     

The Role of Gravity in Apical Dominance : Effects of Clinostating on Shoot Inversion-Induced Ethylene Production, Shoot Elongation, and Lateral Bud Growth

Shoot inversion-induced release of apical dominance in Pharbitis nil is inhibited by rotating the plant at 0.42 revolutions per minute in a vertical plane perpendicular to the axis of rotation of a horizontal clinostat. Clinostating prevented lateral bud outgrowth, apparently by negating the restriction of the shoot elongation via reduction of ethylene production in the inverted shoot. Radial stem expansion was also decreased. Data from experiments with intact tissue and isolated segments indicated that shoot-inversion stimulates ethylene production by increasing the activity of 1-aminocyclopropane-1-carboxylic acid synthase. The results support the hypothesis that shoot inversion-induced release of apical dominance in Pharbitis nil is due to gravity stress and is mediated by ethylene-induced retardation of the elongation of the inverted shoot.     

The effect of uni-axial clinostat rotation on germination and root anatomy of Phaseolus vulgaris L.

ABSTRACT

Phaseolus vulgaris L. seed germination and seedling root anatomy were investigated on a slowly rotating clinostat in 1g. Clinostat rotating seeds were oriented as follows: the first group with the longer axis parallel to the rotation pole (horizontal), the other with the longer axis normal to the rotation pole with due attention to the position of the root apex primordium in the dry seeds (vertical). Germination time, percent germination and curvature of developing roots were monitored. Furthermore, the anatomy of the root apex was quantitatively analysed. Seeds placed on the clinostat germinated earlier than controls, and columella cells of roots developed while rotating lost the strict polarity with the nucleus positioned near the proximal periclinal cell wall and amyloplasts sedimented on the distal periclinal wall. Irrespective of seed orientation on the rotation axis, loss of cell polarity occurred as well as a decrease in starch content, modification in cell size, and damage to statocytes whose walls appeared partially digested. Cell size in the elongation zone was also larger in roots rotating on the clinostat than in controls, both in vertically and horizontally placed specimens. Our results demonstrate that prolonged rotation has an effect on the statocyte that continuously perceives gravity from ever-changing directions, although this effect is irrespective of seed position on the rotating axis in P. vulgaris.     

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.     

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.     

Increased Ethylene Production during Clinostat Experiments May Cause Leaf Epinasty

Ethylene production from tomato (Lycopersicum esculentum L. cv. Rutgers) plants based on a clinostat doubled during the first 2 hours of rotation. Carbon dioxide blocked the appearance of leaf epinasty normally associated with plants rotated on a clinostat. These results support the idea that epinasty of clinostated plants was due to increased ethylene production and not to the cancellation of the gravitational pull on auxin transport in the petiole.     

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.     

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.     

Autonomic Straightening after Gravitropic Curvature of Cress Roots

Few studies have documented the response of gravitropically curved organs to a withdrawal of a constant gravitational stimulus. The effects of stimulus withdrawal on gravitropic curvature were studied by following individual roots of cress (Lepidium sativum L.) through reorientation and clinostat rotation. Roots turned to the horizontal curved down 62° and 88° after 1 and 5 h, respectively. Subsequent rotation on a clinostat for 6 h resulted in root straightening through a loss of gravitropic curvature in older regions and through new growth becoming aligned closer to the prestimulus vertical. However, these roots did not return completely to the prestimulus vertical, indicating the retention of some gravitropic response. Clinostat rotation shifted the mean root angle −36° closer to the prestimulus vertical, regardless of the duration of prior horizontal stimulation. Control roots (no horizontal stimulation) were slanted at various angles after clinostat rotation. These findings indicate that gravitropic curvature is not necessarily permanent, and that the root retains some commitment to its equilibrium orientation prior to gravitropic stimulation.     

Analysis of peg formation in cucumber seedlings grown on clinostats and in a microgravity (space) environment.

In young cucumber seedlings, the peg is a polar out-growth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. Previous studies have indicated that peg formation is gravity dependent. In this study we analyzed peg formation in cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS 95) for 1-2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space grown seedlings was found to be nearly identical to that of clinostat grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; approximately 6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space grown seedlings, compared to clinostat grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground grown seedlings.     

Role of Indole-3-acetic Acid in Modification of Geotropic Responses in Clinostat Rotated Avena Seedlings

Oat seedlings were grown in a sand medium on clinostats with horizontal axes of rotation to nullify the directional component of the gravity-force vector. Coleoptile segments from such seedlings showed an enhanced absorption of apically applied exogenous auxin (indole-3-acetic acid), compared to segments from vertically rotated or stationary controls. Absorption of basally applied auxin and auxin transport were unaffected by the gravity treatments. Horizontal rotation did not materially change the amount of auxin produced and transported from excised coleoptile tips: however, plants so rotated showed an enhanced curvature response to unilaterally applied auxin.

Collectively, these experiments indicate that enhanced plant responses to horizontal clinostat rotation, where rates of rotation are sufficient to nullify the directional component of the gravity-force vector, are caused primarily by increases in metabolism and not by a modification of auxin availability. These data do not support recently advanced hypotheses that the polarity of auxin transport is based on gravitational sedimentation of cell inclusions.