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How to detect when cells in space perceive gravity

It is useful to be able to measure when and whether cells detect gravity during spaceflights. For studying gravitational physiology, gravity perception is the response the experimentalist needs to measure. Also, for growing plants in space, plant cells may have a non-directional requirement for gravity as a development cue. The main goals of spaceflight experiments in which gravity perception would be measured are to determine the properties of the gravity receptor and how it is activated, and to determine fundamental characteristics of the signal generated. The main practical difficulty with measuring gravity sensing in space is that gravity sensing cannot be measured with certainty on earth. Almost all experiments measure gravitropic curvature. Reciprocity and intermittent stimulation are measurements which were made to some degree on earth using clinostatting, but which would provide clearer results if done with microgravity rather than clinostatting. These would be important uses of the space laboratory for determining the nature of gravity sensing in plants. Those techniques which do not use gravitropic curvature to measure gravity sensing are electrophysiological. The vibrating probe would be somewhat easier to adapt to space conditions than the intracellular microelectrode because it can be positioned with less precision. Ideally, a non-invasive technique would be best suited if an appropriate measure could be developed. Thus, the effect of microgravity on cultured cells is more likely to be by large-scale physical events than gravity sensing in the culture cells. It is not expected that it will be necessary to determine whether individual cultured cells perceive gravity unless cells grow abnormally even after the obvious microgravity effects on the culture as a whole can be ruled out as the cause.

Bjoerkman, Thomas

Curvature in Arabidopsis inflorescence stems is limited to the region of amyloplast displacement

Gravitropic sensing in stems and stem-like organs is hypothesized to occur in the endodermis. However, since the endodermis runs the entire length of the stem, the precise site of gravisensing has been difficult to define. In this investigation of gravisensitivity in inflorescence stems of Arabidopsis, we positioned stems in a high gradient magnetic field (HGMF) on a rotating clinostat. Approximately 40% of the young, wild-type (WT) inflorescences, for all positions tested, curved toward the HGMF in the vicinity of the stem exposed to the field. In contrast, when the wedge was placed in the basal region of older inflorescence stems, no curvature was observed. As a control, the HGMF was applied to a starchless mutant, and 5% of the stems curved toward the field. Microscopy of the endodermis in the WT showed amyloplast displacement in the vicinity of the HGMF. Additional structural studies demonstrated that the basal region of WT stems experienced amyloplast displacement and, therefore, suggest this region is capable of gravity perception. However, increased lignification likely prevented curvature in the basal region. The lack of apical curvature after basal amyloplast displacement indicates that gravity perception in the base is not transmitted to the apex. Thus, these results provide evidence that the signal (and thus, response) resulting from perception in Arabidopsis inflorescence stems is spatially restricted.

NASA Discipline Plant Biology

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185-197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.

NASA Discipline Plant Biology

Light regulation of the growth response in corn root gravitropism

Roots of Merit variety corn (Zea mays L.) require red light for orthogravitropic curvature. Experiments were undertaken to identify the step in the pathway from gravity perception to asymmetric growth on which light may act. Red light was effective in inducing gravitropism whether it was supplied concomitant with or as long as 30 minutes after the gravity stimulus (GS). The presentation time was the same whether the GS was supplied in red light or in darkness. Red light given before the GS slightly enhanced the rate of curvature but had little effect on the lag time or on the final curvature. This enhancement was expanded by a delay between the red light pulse and the GS. These results indicate that gravity perception and at least the initial transduction steps proceed in the dark. Light may regulate the final growth (motor) phase of gravitropism. The time required for full expression of the light enhancement of curvature is consistent with its involvement in some light-stimulated biosynthetic event.

NASA Discipline Number 40-50

The response to gravity is correlated with the number of statoliths in Chara rhizoids

In contrast to higher plants, Chara rhizoids have single membrane-bound compartments that appear to function as statoliths. Rhizoids were generated by germinating zygotes of Chara in either soil water (SW) medium or artificial pond water (APW) medium. Differential-interference-contrast microscopy demonstrated that rhizoids form SW-grown plants typically contain 50 to 60 statoliths per cell, whereas rhizoids from APW-grown plants contain 5 to 10 statoliths per cell. Rhizoids from SW are more responsive to gravity than rhizoids from APW because (a) SW rhizoids were oriented to gravity during vertical growth, whereas APW rhizoids were relatively disoriented, and (b) curvature of SW rhizoids was 3 to 4 times greater throughout the time course of curvature. The growth rate of APW rhizoids was significantly greater than that of SW-grown rhizoids. This latter result suggests that APW rhizoids are not limited in their ability for gravitropic curvature by growth and that these rhizoids are impaired in the early stages of gravitropism (i.e. gravity perception). Plants grown in APW appeared to be healthy because of their growth rate and the vigorous cytoplasmic streaming observed in the rhizoids. This study is comparable to earlier studies of gravitropism in starch-deficient mutants of higher plants and provides support for the role of statoliths in gravity perception.

Non-NASA Center

Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis

Gravitropism was examined in dark- and light-grown hypocotyls of wild-type (WT), two reduced starch mutants (ACG 20 and ACG 27), and a starchless mutant (ACG 21) of Arabidopsis. In addition, the starch content of these four strains was studied with light and electron microscopy. Based on time course of curvature and orientation studies, the graviresponse in hypocotyls is proportional to the amount of starch in a genotype. Furthermore, starch mutations seem to primarily affect gravitropism rather than differential growth since both phototropic curvature and growth rates among the four genotypes are approximately equal. Our results suggest that gravity perception may require a greater plastid mass in hypocotyls compared to roots. The kinetics of gravitropic curvature also was compared following reorientation at 45 degrees, 90 degrees, and 135 degrees. As has been reported for other plant species, the optimal angle of reorientation is 135 degrees for WT Arabidopsis and the two reduced starch mutants, but the magnitude of curvature of the starchless mutant appears to be independent of the initial angle of displacement. Taken together, the results of the present study and our previous experiments with roots of the same four genotypes [Kiss et al. (1996) Physiol. Plant. 97: 237] support a plastid-based hypothesis for gravity perception in plants.

Non-NASA Center

Plant biology in space: proceedings of the International Workshop, Bad Honnef, Germany, June 24-27, 1996

Papers presented at the International Workshop on Plant Biology in Space include reviews, reports, and perspectives related to plant gravitational biology. Presentations focused on nine subject areas: gravitropism in unicellular plants, gravitropism in fungi, cell development, gravity perception in multicellular plants, gravity responses in multicellular plants, plant reproduction, evaluation of a clinostat for weightlessness simulation, biological life support systems, and future research.

NASA Discipline Plant Biology

Transduction of the Root Gravitropic Stimulus: Can Apical Calcium Regulate Auxin Distribution?

The hypothesis was tested that calcium, asymmetrically distributes in the root cap upon reorientation to gravity, affects auxin transport and thereby auxin distribution at the elongation zone. It is assumed that calcium exists in the root cap and is asymmetrically transported in root caps altered from a vertical to a horizontal position and that the meristem, the tissue immediately adjacent to the root cap and lying between the site of gravity perception and the site of gravity response, is essential for mediation of gravitropism. Tip calcium in root gravicurvature was implicated. The capstone evidence is that the root cap has the capacity to polarly translocate exogenous calcium downward when tissue is oriented horizontally, and that exogenous calcium, when supplied asymmetrically at the root tip, induces curvature and dictates the direction of curvature in both vertical and horizontal corn roots.

Edwards, K. L.

Protein and carotenoid synthesis and turnover in gravistimulated root caps

In certain cultivars of corn gravitropic bending occurs only after the root cap, the site of gravity perception, is exposed to light. Light appears to trigger or to remove some block in the gravity translation process. Using light sensitive cultivars of corn, it was shown that light affects various processes in the cap. The roles of these light-induced processes in gravitropic bending in roots were studied.

Feldman, L. J.

Modulation of statolith mass and grouping in white clover (Trifolium repens) growth in 1-g, microgravity and on the clinostat

Current models of gravity perception in higher plants focus on the buoyant weight of starch-filled amyloplasts as the initial gravity signal susceptor (statolith). However, no tests have yet determined if statolith mass is regulated to increase or decrease gravity stimulus to the plant. To this end, the root caps of white clover (Trifolium repens) grown in three gravity environments with three different levels of gravity stimulation have been examined: (i) 1-g control with normal static gravistimulation, (ii) on a slow clinostat with constant gravistimulation, and (iii) in the stimulus-free microgravity aboard the Space Shuttle. Seedlings were germinated and grown in the BioServe Fluid Processing Apparatus and root cap structure was examined at both light and electron microscopic levels, including three-dimensional cell reconstruction from serial sections. Quantitative analysis of the electron micrographs demonstrated that the starch content of amyloplasts varied with seedling age but not gravity condition. It was also discovered that, unlike in starch storage amyloplasts, all of the starch granules of statolith amyloplasts were encompassed by a fine filamentous, ribosome-excluding matrix. From light micrographic 3-D cell reconstructions, the absolute volume, number, and positional relationships between amyloplasts showed (i) that individual amyloplast volume increased in microgravity but remained constant in seedlings grown for up to three days on the clinostat, (ii) the number of amyloplasts per cell remained unchanged in microgravity but decreased on the clinostat, and (iii) the three-dimensional positions of amyloplasts were not random. Instead amyloplasts in microgravity were grouped near the cell centers while those from the clinostat appeared more dispersed. Taken together, these observations suggest that changing gravity stimulation can elicit feedback control over statolith mass by changing the size, number, and grouping of amyloplasts. These results support the starch-statolith theory of graviperception in higher plants and add to current models with a new feedback control loop as a mechanism for modulation of statolith responsiveness to inertial acceleration.

Flight Experiment

NASA space biology accomplishments, 1983-84

Approximately 42 project summaries from NASA's Space Biology Program are presented. Emphasis is placed on gravitational effects on plant and animal life. The identification of gravity perception; the effects of weightlessness on genetic integrity, cellular differentiation, reproduction, development, growth, maturation, and senescence; and how gravity affects and controls physiology, morphology, and behavior of organisms are studied.

Halstead, T. W.

Gravity receptors in a microcrustacean water flea - Sensitivity of antennal-socket setae in Daphnia magna

Gravity receptors of Dephnia magna were discovered on the basal segment of the swimming antennae and were shown to respond to upward water currents that pass the animal as it sinks between swimming strokes. Sensitivity of the gravity perceiving mechanism was tested by subjecting daphnids to a series of five decreasingly dense aqueous solutions (neutral density to water) in darkness (to avoid visual cues). Three-dimensional, video analysis of body position (pitch, yaw and roll) and swimming path (hop and sink, vertical and horizontal patterns) revealed a gradual threshold that occurred near a density difference between the animal and its environment of less than 0.25%. Because daphnids do not sink but continue to slide after stroking in the increased density solutions, gravity perception appears to occur during a vertical swing of the longitudinal body axis to the vertical plane, about their center of gravity, and, thereby, implies a multidirectional sensitivity for the antennal-socket setae.

Meyers, D. G.

Advances in space biology and medicine. Vol. 1

Topics discussed include the effects of prolonged spaceflights on the human body; skeletal responses to spaceflight; gravity effects on reproduction, development, and aging; neurovestibular physiology in fish; and gravity perception and circumnutation in plants. Attention is also given to the development of higher plants under altered gravitational conditions; the techniques, findings, and theory concerning gravity effects on single cells; protein crystal growth in space; and facilities for animal research in space.

Bonting, Sjoerd L.

Mining the Gravity Mutants of Arabidopsis

Gravity mutants are a valuable resource for understanding gravity perception, signaling, and response in plants. A review of 190 publications resulted in a list of 97 loci with mutations that caused a gravitropic phenotype. While all these mutants show some form of gravity phenotype, several are also generally defective in growth. After removing these nonspecific mutants, 76 loci were deemed to have true gravity mutants. The gene list was then used to create a sortable database containing key factors of gravity signaling as well as data on methodologies and the development of mutant lines. Indexing this database has allowed us to pull out trends that were not visible in individual publications. For example, mutants that are unable to rearrange starch statoliths make up a larger proportion of the described inflorescence stems mutants than any other organ. Comparing these statolith mutants across organs shows that root and inflorescence stems consistently display different phenotypes for the same class of defect. The more severe gravity defects are most commonly described in root tissue while other tissues are more likely to show only a delayed or reduced response to gravity. The experience of members of the GeneLab plant Analytics Working Group (AWG) in data visualization and gene mapping have sparked new ideas for utilizing this database. Knowledge of shared traits, mutant development, and growth can provide a new resource for the production of seed lines specialized for their response to gravity.

gravitropism

Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight

The "starch-statolith" hypothesis has been used by plant physiologists to explain the gravity perception mechanism in higher plants. In order to help resolve some of the controversy associated with ground-based research that has supported this theory, we performed a spaceflight experiment during the January 1997 mission of the Space Shuttle STS-81. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then given a gravity stimulus on a centrifuge. In terms of development in space, germination was greater than 90% for seeds in microgravity, and flight seedlings were smaller (60% in total length) compared to control plants grown on the ground and to control plants on a rotating clinostat. Seedlings grown in space had two structural features that distinguished them from the controls: a greater density of root hairs and an anomalous hypocotyl hook structure. However, the slower growth and morphological changes observed in the flight seedlings may be due to the effects of ethylene present in the spacecraft. Nevertheless, during the flight hypocotyls of WT seedlings responded to a unilateral 60 min stimulus provided by a 1-g centrifuge while those of the starch-deficient strains did not. Thus the strain with the greatest amount of starch responded to the stimulus given in flight and therefore, these data support the starch-statolith model for gravity sensing.

short duration

Gravitropism in roots of intermediate-starch mutants of Arabidopsis

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

NASA Program Space Biology

Gravity-regulated differential auxin transport from columella to lateral root cap cells

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.

Non-NASA Center

A down to earth model of gravisensing or Newton's Law of Gravitation from the apple's perspective

The physiology of gravity perception in plants is examined and a model of gravitational pressure is explained and compared to the statolith model. The gravitational pressure model is based on studies of tension and compression of the plasma membrane against the extracellular matrix. Further studies examine the role of peptides or enzymes that inhibit a compression receptor and calcium channels.

Non-NASA Center