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.

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.

The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient.

The actin cytoskeleton has been implicated in regulating plant gravitropism. However, its precise role in this process remains uncertain. We have shown previously that disruption of the actin cytoskeleton with Latrunculin B (Lat B) strongly promoted gravitropism in maize roots. These effects were most evident on a clinostat as curvature that would exceed 90 degrees despite short periods of horizontal stimulation. To probe further the cellular mechanisms underlying these enhanced gravity responses, we extended our studies to roots of Arabidopsis. Similar to our observations in other plant species, Lat B enhanced the response of Arabidopsis roots to gravity. Lat B (100 nm) and a stimulation time of 5-10 min were sufficient to induce enhanced bending responses during clinorotation. Lat B (100 nm) disrupted the fine actin filament network in different regions of the root and altered the dynamics of amyloplasts in the columella but did not inhibit the gravity-induced alkalinization of the columella cytoplasm. However, the duration of the alkalinization response during continuous gravistimulation was extended in Lat B-treated roots. Indirect visualization of auxin redistribution using the DR5:beta-glucuronidase (DR5:GUS) auxin-responsive reporter showed that the enhanced curvature of Lat B-treated roots during clinorotation was accompanied by a persistent lateral auxin gradient. Blocking the gravity-induced alkalinization of the columella cytoplasm with caged protons reduced Lat B-induced curvature and the development of the lateral auxin gradient. Our data indicate that the actin cytoskeleton is unnecessary for the initial perception of gravity but likely acts to downregulate gravitropism by continuously resetting the gravitropic-signaling system.

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).

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.

Mechanotransduction in gravisensing cells.

Plant organs can re-orientate themselves with respect to gravity. Gravisensing cells (statocytes) contain movable amyloplasts whose potential energy is apparently used to activate calcium channels by exerting tension on the actin network and/or pressure on the cytoskeleton elements lining the plasma membrane. The chain of events that follows remains to be further analysed but includes transient pH changes in the cytosol and sustained pH changes in the cell wall. Transduction ends with relocation of the auxin efflux carriers responsible for the lateral transport of auxin, which reorients the root tip in the direction of gravity. Many questions remain to be solved but recent studies now herald a better understanding of the molecular events involved in gravisensing.

Development of the primary root and mobilisation of reserves in etiolated seedlings of Brassica napus grown on a slowly rotating clinostat.

The effect of the slow rotating clinostat (1 rpm) on the growth of the primary root was studied on Brassica napus seedlings. After 5 d in darkness, the primary root was longer and thinner in seedlings grown on the clinostat than in seedlings grown in the vertical position. However, the breakdown of lipid reserves, sucrose level and transport of 14C-labeled sucrose from the cotyledons to the primary root, were not altered by growth on the clinostat. Moreover, the activity of isocitrate lyase, one of the two enzymes necessary for the conversion of lipids into glucids also was also not modified in the cotyledons of clinorotated seedlings. Thus, there was clear evidence that clinorotation had a direct effect on the growth of the primary root that was independent of the mobilisation of lipid reserves in the cotyledons. As a sink, the primary root had the same strength on the clinostat as in the vertical position, but the reserves were used in a different way. The increase in root elongation on the clinostat could be due to the slight, but continuous, omnilateral gravitropic stimulation due to the rotation of the seedlings about a horizontal axis.

The dose-response curve of the gravitropic reaction: a re-analysis.

The dose-response curve of the gravitropic reaction is often used to evaluate the gravisensing of plant organs. It has been proposed (Larsen 1957) that the response (curvature) varies linearly as a function of the logarithm of the dose of gravistimulus. As this model fitted correctly most of the data obtained in the literature, the presentation time (tp, minimal duration of stimulation in the gravitational field to induce a response) or the presentation dose (dp, minimal quantity in g.s of stimulation to induce a response) were estimated by extrapolating down to zero curvature the straight line representing the response as a function of the logarithm of the stimulus. This method was preferred to a direct measurement of dp or tp with minute stimulations, since very slight gravitropic response cannot be distinguished from the background oscillations of the extremity of the organs. In the present review, it is shown that generally the logarithmic model (L) does not fit the experimental data published in the literature as well as the hyperbolic model (H). The H model in its simplest form is related to a response in which a ligand-receptor system is the limiting phase in the cascade of events leading to the response (Weyers et al. 1987). However, it is demonstrated that the differential growth, responsible for the curvature (and the angle of curvature), would vary as a hyperbolic function of the dose of stimulation, even if several steps involving ligand-receptor systems are responsible for the gravitropic curvature. In the H model, there is theoretically no presentation time (or presentation dose) since the curve passes through the origin. The value of the derivative of the H function equals a/b and represents the slope of the cune at the origin. It could be therefore used to estimate gravisensitivity. This provides a measurement of graviresponsiveness for threshold doses of stimulation. These results imply that the presentation time (or presentation dose) derived from the L model cannot be used anymore as an estimate of gravisensitivity. On the contrary, the perception time (minimal duration of a repeated stimulation which induces a response), which is less than 1 s, should be related to the perception of gravity. The consequences of these results on the mode of action and the nature of graviperception are discussed.

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.