Root Statocytes Research Articles

Statoliths displacement in root statocytes in real and simulated microgravity

Despite the long-term employment of different types of clinostats in space and gravitational biology, the discussions about their reliability to mimic microgravity in space flight are still ongoing. In this paper, we present some data about the behaviour of amyloplasts-statoliths in root cap statocytes of higher plant seedlings growing during 3–5 days under slow and fast 2-D clinorotation and real microgravity in orbital flight. In addition, data on the displacement of amyloplasts in the statocytes of seedlings subjected to vibration and acceleration in the launch mode of a spacecraft are also given. A comparative analysis showed sharp differences in statolith responses to slow and fast clinorotation with a speed of 50 rpm. In the first case, the behaviour of amyloplasts was more or less similar to that in space flight, they did not touch the plasmalemma. In the second case, the contacts of statoliths with the plasmalemma or its invaginations (plasmalomasomes), like those under the action of vibration and acceleration, were clearly observed. Thus, slow 2-D clinostat is more suitable to study gravity sensing by root cap amyloplasts-statoliths and their responses to microgravity in the ground-based experiments.

Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity

Gravity is a permanent environmental signal guiding plant growth and development. Gravity sensing in plants starts with the displacement of starch-filled plastids called statoliths, ultimately leading to auxin redistribution and organ curvature. While the involvement in gravity sensing of several actors such as calcium is known, the effect of statolith displacement on calcium changes remains enigmatic. Microgravity is a unique environmental condition offering the opportunity to decipher this link. In this study, roots of Brassica napus were grown aboard the International Space Station (ISS) either in microgravity or in a centrifuge simulating Earth gravity. The impact of short simulated gravity onset and removal was measured on statolith positioning and intracellular free calcium was assessed using pyroantimonate precipitates as cytosolic calcium markers. Our findings show that a ten-minute onset or removal of gravity induces very low statolith displacement, but which is, nevertheless, associated with an increase of the number of pyroantimonate precipitates. These results highlight that a change in the cytosolic calcium distribution is triggered in absence of a significant statolith displacement.

Mechanism of Higher Plant Gravity Sensing

Higher plants have developed statocytes, specialized tissues or cells for gravity sensing, and subsequent signal formation. Root and shoot statocytes commonly harbor a number of amyloplasts, and amyloplast sedimentation in the direction of gravity is a critical process in gravity sensing. However, the molecular mechanism underlying amyloplast-dependent gravity sensing is largely unknown. In this review, we mainly describe the molecular basis for the gravity sensing mechanism, i.e., the molecules and their functions involved in amyloplast sedimentation. Several analyses of statocyte images in living plant organs have implied differences in the regulation of amyloplast movements between root and shoot statocytes. Amyloplasts in shoot statocytes display not only sedimentable but upward, saltatory movements, but the latter are rarely observed in root statocytes. A series of genetic studies on shoot gravitropism mutants of Arabidopsis thaliana has revealed that two intracellular components, the vacuolar membrane (VM) and actin microfilaments (AFs), within the shoot statocyte play important roles in amyloplast dynamics. Flexible VM structures surrounding the amyloplasts seem to allow them to freely sediment toward the bottom of cells. In contrast, long actin cables mediate the saltatory movements of amyloplasts. Thus, amyloplasts in shoot statocytes undergo a dynamic equilibrium of movement, and a proper intracellular environment for statocytes is essential for normal shoot gravitropism. Further analyses to identify the molecular regulators of amyloplast dynamics, including sedimentation, may contribute to an understanding of the gravity sensing mechanism in higher plants.

Comparison Study of Gravity-Dependent Displacement of Amyloplasts in Statocytes of Cress Roots and Hypocotyls

In this study, the kinetics of gravity-dependent movement of amyloplasts (statoliths) along root statocytes and hypocotyls (endodermis cells) has been analyzed and compared in order to testify cytoskeleton involvement in the displacement of statoliths in cress (Lepidium sativum L.) seedling statocytes. After 32 h of growth at 1 g or under a fast clinorotation (50 rpm), the seedlings were treated for 24 min as follows: exposition to clinorotation or 180° inversion and the action of gravitational force in root tip or hypocotyl tip direction. Statolith displacement was studied by light microscopy on semi-thin longitudinal sections of hypocotyls and root caps, measuring the distance between the centre of plastids and morphological cell bottom. Considerable temporal differences have been determined between the kinetics of the longitudinal motion of amyloplasts in root and hypocotyl statocytes of 1-g seedlings upon exposition to fast clinorotation and inversion. In statocytes of both organs of seedlings grown under fast clinorotation, the gravity provoked displacement of statoliths in the direction of its action; however, the displacement was much faster in hypocotyl than in root statocytes. It has been assumed that the gravity-determined longitudinal transport of amyloplasts, both in hypocotyl endodermic cells and root statocytes of cress seedlings, is modulated by the cytoskeleton.

Do ARG1 and ARL2 form an actin-based gravity-signaling chaperone complex in root statocytes?

Plants are acutely sensitive to the directional information provided by gravity. They have evolved statocytes, which are specialized cells that sense gravity and, upon integration of the corresponding information with that of other environmental stimuli, control the growth behavior of their organs. The cellular mechanisms that allow statocytes to sense and transduce gravitational information likely involve detecting the sedimentation of, or the tension/pressure exerted by, starch-filled amyloplasts - the presumptive statoliths - within their cytoplasm. Gravity signaling in root statocytes controls the direction of transport of signaling compounds, especially auxin, across the root cap, establishing a lateral gradient that is transmitted to cells in the elongation zone and results in gravitropic curvature. The Arabidopsis J-domain proteins ARG1 and ARL2 function as gravity-signal transducers in root statocytes. In the January issue of Plant Journal, we reported that ARG1 and ARL2 function non-redundantly in a common gravity signaling pathway required for accumulation of the auxin efflux facilitator PIN3 on the new bottom side of statocytes following gravity stimulation, and lateral redistribution of auxin toward the new lower flank of stimulated roots. Here we present data suggesting that ARG1 physically associates with ARL2, the J-domain co-chaperone HSC70, and actin in vivo. We briefly discuss potential mechanisms by which ARG1 and ARL2 might function in gravity signaling in light of this information.

ARL2, ARG1 and PIN3 define a gravity signal transduction pathway in root statocytes

ALTERED RESPONSE TO GRAVITY1 (ARG1) and its paralog ARG1-LIKE2 (ARL2) are J-domain proteins that are required for normal root and hypocotyl gravitropism. In this paper, we show that both ARL2 and ARG1 function in a gravity signal transduction pathway with PIN3, an auxin efflux facilitator that is expressed in the statocytes. In gravi-stimulated roots, PIN3 relocalizes to the lower side of statocytes, a process that is thought to, in part, drive the asymmetrical redistribution of auxin toward the lower flank of the root. We show that ARL2 and ARG1 are required for PIN3 relocalization and asymmetrical distribution of auxin upon gravi-stimulation. ARL2 is expressed specifically in the root statocytes, where it localizes to the plasma membrane. Upon ectopic expression, ARL2 is also found at the cell plate of dividing cells during cytokinesis, an area of intense membrane dynamics. Mutations in ARL2 and ARG1 also result in auxin-related expansion of the root cap columella, consistent with a role for ARL2 and ARG1 in regulating auxin flux through the root tip. Together these data suggest that ARL2 and ARG1 functionally link gravity sensation in the statocytes to auxin redistribution through the root cap.

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

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.

Adenosine Kinase Modulates Root Gravitropism and Cap Morphogenesis in Arabidopsis

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

Kinetics of gravity-induced amyloplast sedimentation in statocytes of cress roots grown under fast clino-rotation, 1 g and after 180° inversion

In order to detail the relation between the initial positioning of statoliths and root graviresponsiveness, the movement of amyloplasts within root statocytes of cress ( Lepidium sativum L.) seedlings grown under fast clino-rotation (50 rpm) and vertically at 1 g or 180° inverted after growth at 1 g has been analyzed during a subsequent 6-min period of lateral (reoriented 90°) gravistimulation. The main parameters of statolith motion kinetics such as relative positions and motion velocities in transverse and longitudinal directions have been evaluated taking linear measurements of organelle position with respect to the lower longitudinal and distal wall in the cells of the 2nd–5th columella storeys. A significant discrepancy in the direction and velocity of statolith movement has been determined within the first and second minutes of root horizontal placement. Centrally or proximally located statoliths in clino-rotated or inverted root statocytes moved transversely to the lower longitudinal cell wall more quickly as compared with distally located ones in 1- g root cells. At the same time, the longitudinal displacement of proximally located statoliths towards the cell center has been determined, while this displacement of distally located plastids was significantly more pronounced. The longitudinal motion of amyloplasts grouped in the central cell part was negligible within this 2-min period. After the 6th minute of stimulation, the total downward shift of the mean relative position of statoliths amounted to 16.3% from the total cell width in clino-rotated root statocytes and 17.1% in the cells of inverted roots, while it equalled 13.3% in 1- g root statocytes. The total change of the relative longitudinal position of amyloplasts with respect to the distal cell wall remained almost unchanged in roots grown on the clinostat. In inverted roots, the initial longitudinal position of 56.2% from the total cell length has been shifted significantly to 47.8% and from 27.7% to 38.8% in 1- g roots. The data imply that a more pronounced sedimentation of centrally or proximally located amyloplasts during the first minutes of root stimulation could be responsible for the earlier generation and transmission of a directional signal which triggers the subsequent events of gravitropic reaction in clino-rotated or inverted roots as compared to 1- g roots.

Action of the mechanical disruption of the actin network on the gravisensitivity of the root statocyte

The effects of the mechanical disruption of the thin actin network of statocytes on gravisensitivity have been studied on lentil roots. Seedling roots were first inverted for 7 min (root tip upward) and then placed in the downward (normal) position for 7 min before gravitropic stimulation in the horizontal position. The period of inversion allowed the amyloplasts to move from the distal part to the proximal part of the statocyte, but did not fully sediment. When the roots were returned to the tip down position, the amyloplasts moved toward the distal part, but also did not completely sediment by the time the roots were placed horizontally. Thus, in these roots the amyloplasts could be still moving toward the distal wall after they had been replaced in the normal position and the actin network should not be fully restored. Gravisensitivity was estimated by the analysis of the dose–response curves of vertical and treated (inverted and returned to downward position) roots. The only effect, which has been observed on treated roots, was a delay of graviresponse for about 1 min. Our interpretation of this result is that in vertical roots the amyloplasts can exert tensions in the actin network that are directly transmitted to mechanoreceptors located in the plasma membrane. In roots with a partially disrupted actin network, a delay of 1 min is necessary for the amyloplasts to activate mechanoreceptors.

A polarized cell: the root statocyte.

In the gravity-perceiving cells (statocytes), located in the centre of the root cap, polarity is expressed in the arrangement of the organelles since, in most genera, the nucleus and the endoplasmic reticulum are maintained at the opposite ends of each cell by actin. Polarity is also evident in the distribution of plasmodesmata, which are more numerous in the transverse walls than in the longitudinal walls. The centre of each statocyte is depleted of microtubules (they are only located at the periphery) but is occupied by numerous amyloplasts (statoliths), denser than the cytoplasm. The amyloplasts do not contribute to the inherent structural polarity since their position is dependent upon the gravity vector. This article focuses on new microscopic analyses and on data obtained from experiments performed in microgravity, which have contributed to our better understanding of the architecture of the actin web implicated in the perception of gravity. Depending upon the plant, the actin network seems to be formed of single filaments arranged in various ways, or, of thin bundles of actin filaments. The amyloplasts are enmeshed in this web of actin and their envelopes are associated with it, but they can have autonomous movement via myosin in the absence of gravity. From calculations of the value of the force necessary to move one amyloplast in the lentil root, and from videomicroscopy performed with living statocytes of maize roots, it is hypothesized that actin microfilaments could be orientated in an overall diagonal direction in the statocyte. These observations could help in understanding how slight amyloplast movements may trigger and transmit the gravitropic signal.

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 μm/min, which decreased to 0.12 ± 0.01 μm/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° 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 clino-rotation.

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.

An active role of the amyloplasts and nuclei of root statocytes in graviperception.

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception.

Immunolocalization of actin in root statocytes of Lens culinaris L.

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.

Gravisensitivity of cress roots

The minimum dose (stimulus x time [gs]) eliciting a visible gravitropic response, has been determined using continuous and intermittent stimulation and two different accelerations at 1g and 0.1g. The minimum dose of 20 – 30 gs estimated for microgravity roots and of 50 – 60 gs for roots grown on a 1g-centrifuge indicated a higher sensitivity of microgravity roots. Applying intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus free interval of 118 s The curvature of microgravity-grown roots to lateral stimulation by 0.1 g was remarkably smaller than by 1g in spite of the same doses which were applied to the seedlings. Microscopic investigations corresponding to stimulations in the range of the threshold values, demonstrated small displacement (< 2 μm) of statoliths in root statocytes. Accepting the statolith theory, one can conclude that stimulus transformation has to occur in the cytoplasm in close vicinity to the statoliths and that this transformation system was affected during seedling cultivation in microgravity.

Calcium balance in pea root statocytes under both clinorotation and ca 2+ channel blockers' influence

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca 2+ level in pea root statocytes. A rise in [Ca 2+] i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca 2+ channel blockers in the growth medium may rescue a plant from abundance of Ca 2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca 2+ channel blocker (1 μM D600 or nicardipine, 12 hr) causes decreasing the Ca 2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca 2+-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca 2+ channel blockers may act as protectors for plants against rise in [Ca 2+]i. The role for Ca 2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca 2+ balance in plant cells in space flights are discussed.

The RHG Gene Is Involved in Root and Hypocotyl Gravitropism in Arabidopsis thaliana

In higher plants, shoots show negative gravitropism and roots show positive gravitropism. To elucidate the molecular mechanisms of root and hypocotyl gravitropism, we segregated the second mutation from the original phyB-1 mutant line which impaired both root and hypocotyl gravitropism and characterized this novel mutation named rhg (for root and hypocotyl gravitropism). The rhg is a single recessive nuclear mutation and it is mapped on the lower part of the chromosome 1. Analyses on the gravitropic responses of the rhg mutant indicate that root and hypocotyl gravitropism are severely impaired but inflorescence stem gravitropism is not affected by the rhg mutation. In the rhg mutant seedlings, amyloplasts (statoliths for gravity-perception) were present in the presumptive statocytes of roots and hypocotyls. Phototropism by roots and hypocotyls was not impaired in the rhg mutant. These results suggest that the RHG gene product probably acts on the gravity-perception and/or the gravity-signal transduction in root and hypocotyl gravitropism. This is the first report about the genetic locus specifically involved in both root and hypocotyl gravitropism but not inflorescence stem gravitropism, supporting our hypothesis that the mechanisms of gravitropism are genetically different between hypocotyls and inflorescence stems.

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.

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 × 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 × 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.