Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Root growth and statocyte polarity in lentil seedling roots grown in microgravity or on a slowly rotating clinostat

The ability of clinostats to simulate microgravity was evaluated by comparing lentil ( Lens culinnrias L. cv. Verte du Puy) seedlings grown in space (Spacelab D1 Mission) with seedlings grown on a slowly rotating elinostat. Seeds were germinated and incubated for 25.5 h at 22°C (1) in microgravity, (2) on a 1g-centrifuge in space. (3) on a slowly rotating elinostat and (4) on the ground. Morphological (root length and orientation) and ultrastructural (distribution of amyloplasts, location of the nucleus in statocytes) parameters were studied. For clinostat experiments, two different configurations were employed: the longitudinal axis of the root was parallel (horizontal elinorotation) or perpendicular (vertical elinorotation) to the axis of rotation. the same configurations were used for the lg-controls. Root length and orientation were similar for roots grown on the clinostat and in microgravity. The amyloplasts were identically distributed in statocytes of horizontally clinorolated roots and in statocytes differentiated in microgravity. However, the location of the nucleus was similar in vertically rotated roots and microgravity samples. Since the involvement of the nucleus in graviperception is not known, it can be concluded that horizontal clinorotation simulates microgravity better than vertical elinorotation.     

The Role of Starch in the Gravitropic Response of the Lentil Root

It is well accepted that the amyloplasts of the cap are responsible for gravisensing in primary roots. However, roots with starch-depleted plastids are able to respond to gravistimulus, but their curvature is slower than that of roots containing amyloplasts. The goal of our experiment was to analyse the effects of natural variations of statolith starch in the gravitropic response of lentil roots to a stimulation in the horizontal position. In lentil seedlings grown in the vertical position for 26 h, the volume of the amyloplasts in the statocytes differed between individual roots. The amount of starch in the cap was determined parallel to the rate of gravitropic curvature. There was no statistical correlation between the intensity of the gravitropic response and the starch content in the statocytes. Lentil roots were treated with gibberellic acid (GA₃) at 32°C in order to reduce the volume of starch in the statoliths. There was 53% less starch in the cap of GA₃treated roots as compared to the cap of control roots. But there was no relationship between starch content in the cap and the responsiveness of the root to a gravistimulus, except when the amount of starch was small.     

Mechanotransduction in root gravity sensing cells

The analysis of the dose-response curve of the gravitropic reaction of lentil seedling roots has shown that these organs are more sensitive when they have been grown in microgravity than when they have been grown on a 1 g centrifuge in space before gravistimulation. This difference of gravisensitivity is not due to the volume or the density of starch grains of statoliths, which are about the same in both conditions (1 g or microgravity). However, the distribution of statoliths within the statocyte may be responsible for this differential sensitivity, since the dispersion of these organelles is greater in microgravity than in 1 g. When lentil roots grown in microgravity or in 1 g are stimulated at 0.93 g for 22 min, the amyloplasts sediment following two different trajectories. They move from the proximal half of the statocytes toward the lower longitudinal wall in the microgravity grown sample and from the distal half toward the longitudinal wall in the 1 g grown sample. At the end of the stimulation, they reach a similar position within the statocytes. If the roots of both samples are left in microgravity for 3 h, the amyloplasts move toward the cell centre in a direction that makes an average angle of 40 degrees with respect to the lower longitudinal wall. The actin filaments, which are responsible for this movement, may have an overall orientation of 40 degrees with respect to this wall. Thus, when roots grown in microgravity are stimulated on the minicentrifuge the amyloplasts slide on the actin filaments, whereas they move perpendicular to them in 1 g grown roots. Our results suggest that greater sensitivity of seedling roots grown in microgravity should be due to greater dispersion of statoliths, to better contacts between statoliths and the actin network and to greater number of activated mechanoreceptors. One can hypothesize that stretch activated ion channels (SACs) located in the plasma membrane are responsible for the transduction of gravistimulus. These SACs may be connected together by elements of the cytoskeleton lining the plasma membrane and to the actin filaments. They could be stimulated by the action of statoliths on the actin network and/or on these elements of the cytoskeleton which link the mechanoreceptors (SACs).     

Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity

The location of the nucleus in statocytes of lentil roots grown: I), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots grown on the clinostat there was a distance of 0.47 μm horizontal clinorotation) and of 0.76 μm vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 μm from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.     

Contributions of space experiments to the study of gravitropism

The study of gravitropism in space has permitted the discovery that statoliths are not completely free to sediment in the gravisensing cells of roots. These organelles are attached to actin filaments via motor proteins (myosin) which are responsible for their displacement from the distal pole of the cell toward the proximal pole when the seedlings are transferred from a 1g centrifuge in space to microgravity. On the ground, the existence of the link between the statoliths and the actin network could not be established because the gravity force is much greater than the force exerted by the motor proteins. This finding led to a new hypothesis on gravisensing. It has been proposed that statoliths can exert tensions in the actin network which become asymmetrical when the root is stimulated in the horizontal position on the ground. The space experiments have confirmed to some extent the results obtained on gravisensitivity with clinostats, although these devices do not simulate microgravity correctly. Reexamination of the means of estimating gravisensitivity has led to the conclusion that the perception and the transduction phases could be very short (that is, within a second). This data is consistent with the fact that the statoliths are attached to the actin filament and do not have to move a long distance to exert forces on the actin network. It has also been demonstrated that gravity regulates the gravitropic bending in order to avoid the overshooting of the vertical direction on the ground. The roots, which are stimulated and placed in microgravity, are not subjected to this regulation and curve more than roots stimulated continuously. However, the curvature of roots or of coleoptiles that takes place in microgravity can be greatly reduced by straightening the extremity of the organs.     

Lentil root statoliths reach a stable state in microgravity

 The kinetics of the movement of statoliths in gravity-perceiving root cap cells of Lens culinaris L. and the force responsible for it have been analysed under 1 g and under microgravity conditions (S/MM-03 mission of Spacehab 1996). At the beginning of the experiment in space, the amyloplasts were grouped at the distal pole of the statocytes by a root-tip-directed 1-g centrifugal acceleration. The seedlings were then placed in microgravity for increasing periods of time (13, 29, 46 or 122 min) and chemically fixed. During the first 29 min of microgravity there were local displacements (mean velocity: 0.154 μm min⁻¹) of some amyloplasts (first at the front of the group and then at the rear). Nevertheless, the group of amyloplasts tended to reconstitute. After 122 min in microgravity the bulk of amyloplasts had almost reached the proximal pole where further movement was blocked by the nucleus. After a longer period in microgravity (4 h; experiment carried out 1994 during the IML 2 mission) the statoliths reached a stable position due to the fact that they were stopped by the nucleus. The position was similar to that observed in roots grown continuously in microgravity. Treatment with cytochalasin D (CD) did not stop the movement of the amyloplasts but slowed down the velocity of their displacement (0.019 μm min⁻¹). Initial movement patterns were the same as in control roots in water. Comparisons of mean velocities of amyloplast movements in roots in space and in inverted roots on earth showed that the force responsible for the movement in microgravity (F_c) was about 86% less (F_c = 0.016 pN) than the gravity force (F_g = 0.11 pN). Treatment with CD reduced F_c by two-thirds. The apparent viscosity of the statocyte cytoplasm was found to be 1 Pa s or 3.3 Pa s for control roots or CD treated roots, respectively. Brownian motion or elastic forces due to endoplasmic reticulum membranes do not cause the movement of the amyloplasts in microgravity. It is concluded that the force transporting the statoliths is caused by the actomyosin system. Received: 22 March 1999 / Accepted: 18 December 1999     

Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity

The location of the nucleus in statocytes or lentil roots grown: 1), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots crown on the clinostat there was a distance of 0.47 micrometers (horizontal clinorotation) and or 0.76 micrometers (vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 micrometers from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.     

Effect of microgravity on the cell cycle in the lentil root

Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the 1-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in 1-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.     

The behaviour of normal and agravitropic transgenic roots of rapeseed (Brassica napus L.) under microgravity conditions

In the TRANSFORM experiment for IML-2 on the Space Shuttle Columbia, normal (wild type = WT) and genetically transformed agravitropic rapeseed roots were tested under microgravity conditions. The aim of the experiment was to determine if the wild-type roots behaved differently (growth, morphology, gravitropical sensitivity) from the transgenic roots. The appearance of the organelles and distribution of statoliths (i.e. amyloplasts with starch grains) in the gravitropic reactive cells (statocytes) under weightlessness was compared for the two types of roots. Attempts have also been made to regenerate new plants from the root material tested in space. Both the WT and the transgenic root types showed the expected increase in length during 36 h of photorecording. Contrary to the results of the ground controls, no significant difference in elongation rates was found between the WT and transgenic roots grown in orbit. However, there are indications that the total growth both in the WT and the transgenic roots was higher in the ground control than for roots in orbit. After a 60 min 1 x g stimulation of the roots on board the Shuttle, no detectable curvatures were obtained in either the transgenic or the WT roots. However, it cannot be excluded that a minute curvature development occurs in the root tips but was not detected due to technical reasons. The ultrastructure was well preserved in both the WT and the transgenic roots, despite the fact that the tissue was kept in the prefixative for over 3 weeks. No marked differences in ultrastructure were observed between the transformed root statocyte cells and the equivalent cells in the wild type. There were no obvious differences in root morphology during the orbital period. Light micrographs and morphometrical analysis indicate that the amyloplasts of both the wild type and transformed root statocytes are randomly distributed over the cells kept under micro-g conditions for 37 h after a 14 h stimulation on the 1 x g centrifuge. The main scientific conclusion from the TRANSFORM experiment is that the difference in growth found in the ground control between the WT and the transgenic root types seems to be eliminated under weightlessness. Explanations for this behaviour cannot be found in the root ultrastructure or in root morphology.     

Relation entre réaction géotropique et évolution du statenchyme dans la racine d'Asperge

Relationship between the Geotropic Response and the Evolution of the Statenchyma in Roots of Asparagus officinalis. The evolution of the statenchyma in roots of Asparagus of ficinalis seedlings, grown in obscurity, was followed during the first 17 days. After 7 days of etiolation, a decrease of both the average diameter of the amyloplasts and the average number of these organelles was observed in the central root cap cells. If the seedlings were illuminated (with white light) from the 7th day, the average number of statoliths increased rapidly in the statocytes. The volume of these organelles undergoes the same variation in etiolated and in illuminated plants. The initial rate of curvature (V_i) of the roots (stimulated in a horizontal position) and the volume of amyloplasts (V_ac) in their caps were analysed as a function of the time of germination in obscurity (from the 8th to the 17th day). It was found that V_i increased as a linear function of the logarithm of V_ac, which confirms that the weight of the amyloplasts of the statocytes may play a role in the geotropic stimulation of the roots.     

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1  g . sgr2 mutants were able to sense and respond to gravity under 30  g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1  g . We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1  g .     

Comparative study of cellular structures implicated in gravisensing in statocytes of primary and lateral roots of Vigna angularis

Summary. The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots. Correspondence and reprints: Department of Biology, Faculty of Science, Ehime University, Bunkyo-cho, Matsuyama 790-8577, Japan.     

Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station

The GRAVI-1 experiment was brought on board the International Space Station by Discovery (December 2006) and carried out in January 2007 in the European Modular Cultivation System facility. For the first run of this experiment, lentil seedlings were hydrated and grown in microgravity for 15 h and then subjected for 13 h 40 min to centrifugal accelerations ranging from 0.29 x 10(-2) g to 0.99 x 10(-2) g. During the second run, seedlings were grown either for 30 h 30 min in microgravity (this sample was the control) or for 21 h 30 min and then subjected to centrifugal accelerations ranging from 1.2 x 10(-2) g to 2.0 x 10(-2) g for 9 h. In both cases, root orientation and root curvature were followed by time-lapse photography. Still images were downlinked in near real time to ground Norwegian User Support and Operations Center during the experiment. The position of the root tip and the root curvature were analyzed as a function of time. It has been shown that in microgravity, the embryonic root curved strongly away from the cotyledons (automorphogenesis) and then straightened out slowly from 17 to 30 h following hydration (autotropism). Because of the autotropic straightening of roots in microgravity, their tip was oriented at an angle close to the optimal angle of curvature (120 degrees -135 degrees ) for a period of 2 h during centrifugation. Moreover, it has been demonstrated that lentil roots grown in microgravity before stimulation were more sensitive than roots grown in 1 g. In these conditions, the threshold acceleration perceived by these organs was found to be between 0 and 2.0 x 10(-3) g and estimated punctually at 1.4 x 10(-5) g by using the hyperbolic model for fitting the experimental data and by assuming that autotropism had no or little impact on the gravitropic response. Gravisensing by statoliths should be possible at such a low level of acceleration because the actomyosin system could provide the necessary work to overcome the activation energy for gravisensing.     

Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity

The minimum dose (dose = stimulus x time), one of three threshold values related to gravity, was determined under microgravity conditions for cress roots. Seedlings were cultivated on a 1g centrifuge in orbit and under microgravity, respectively. After continuous stimulation on a threshold centrifuge, minimum doses of 20-30 gs for microgravity roots and 50-60 gs for roots grown on a 1g centrifuge were estimated, which indicated that microgravity roots have a higher sensitivity than 1g roots. These results do not confirm the threshold value of 12gs which was determined for cress roots using the slow rotating clinostat. Following application of intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus-free interval of 118s. Generally, this demonstrates that higher plants are able to 'sum up' stimuli which are below the threshold value. Microscopic investigations of the cellular structure corresponding to stimulations in the range of the threshold value demonstrated a small displacement of statoliths in root statocytes. No significant correlation was observed between gravitropic curvature and statolith displacement. If the statolith theory is accepted, it can be concluded that stimulus transformation must occur in the cytoplasm in the near vicinity of the statoliths and that this transformation system--probably involving cytoskeletal elements--must have been affected during microgravity seedling cultivation.     

Dorsiventralität der Statocyten in plagiogeotropen Seitenwurzeln von Lepidium sativum L.

Summary The calyptra of plagiotropic lateral roots of Lepidium sativum L. is composed of three rows of cells. Movable amyloplates, possibly functioning as statoliths, are located only a few central cells of the ontogenetic youngest cell row. Beside the lateral root axis the two innermost statocytes contain a stable complex of rough endoplasmic reticulum, which is preferentially located in the central distal cell corner. In the statocytes lying above the lateral root axis the amyloplasts are sedimented on the ER-complex during growth in direction of the geotropic liminal angle. In the statocyte below the axis the ER-complex is free of amyloplasts. Thus a dorsiventrality exists in the statocytes located above and below the root axis in regard to the arrangement of their organelles.

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Herrn Professor Dr. Maximilian Steiner zum 70. Geburtstag.     

Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets

During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Chara globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. 10^–4 · g (i.e. microgravity) during the parabolic flight (lasting for 301–390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 m and 3.6 m in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.Abbreviations ER endoplasmic reticulum - g 9.806 m · s^–2 - MF microfilament - TEXUS Technologische Experimente unter Schwerelosigkeit (technological experiments under reduced gravity) Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthday     

The Influence of Gravity on the Formation of Amyloplasts in Columella Cells of Zea mays L

Columella (i.e., putative graviperceptive) cells of Zea mays seedlings grown in the microgravity of outer space allocate significantly less volume to putative statoliths (amyloplasts) than do columella cells of Earth-grown seedlings. Amyloplasts of flight-grown seedlings are significantly smaller than those of ground controls, as is the average volume of individual starch grains. Similarly, the relative volume of starch in amyloplasts in columella cells of flight-grown seedlings is significantly less than that of Earth-grown seedlings. Microgravity does not significantly alter the volume of columella cells, the average number of amyloplasts per columella cell, or the number of starch grains per amyloplast. These results are discussed relative to the influence of gravity on cellular and organellar structure.     

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.     

Gravity sensing in tip-growing cells

In addition to the statocytes of roots and shoots, a number of tip-growing cells also sense gravity, which influences the cells' growth and development. Since these tip-growing cells are highly suitable for observations in vivo, the movement and sedimentation of their statoliths can be studied in detail. Experimental manipulation by centrifugation, drug application, optical tweezers or microgravity can be monitored by light microscopy. The statoliths are localized in distinct cytoplasmic areas by interactions with actin filaments or microtubules, and their sedimentation seems to be narrowly confined. Since gravisensing and the graviresponse take place within the same cell, the gravitropic signal transduction chain is not complicated by signal transmission between sensing and responding cells. Studies on tip-growing cells have now enabled the formulation of models explaining positive and negative gravitropism.