Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation.     

Basic assumptions and comparison of three gravitropic response models.

Three systemic models of gravitropic response were compared by fitting different experimental data. Results indicate that fits improve with the inclusion of diagravitropic component into formulas that allow to model plagiotropic orientation. More realistic simulation of gravitropic bending along an axial organ does not improve the fit but multiplies the number of parameters and causes correlation among them. However, such a model seems to have more possibilities for further improvement.     

Touch and gravitropic set-point angle interact to modulate gravitropic growth in roots.

Plant roots must sense and respond to a variety of environmental stimuli as they grow through the soil. Touch and gravity represent two of the mechanical signals that roots must integrate to elicit the appropriate root growth patterns and root system architecture. Obstacles such as rocks will impede the general downwardly directed gravitropic growth of the root system and so these soil features must be sensed and this information processed for an appropriate alteration in gravitropic growth to allow the root to avoid the obstruction. We show that primary and lateral roots of Arabidopsis do appear to sense and respond to mechanical barriers placed in their path of growth in a qualitatively similar fashion. Both types of roots exhibited a differential growth response upon contacting the obstacle that directed the main axis of elongation parallel to the barrier. This growth habit was maintained until the obstacle was circumvented, at which point normal gravitropic growth was resumed. Thus, the gravitational set-point angle of the primary and lateral roots prior to encountering the barrier were 95 degrees and 136 degrees respectively and after growing off the end of the obstacle identical set-point angles were reinstated. However, whilst tracking across the barrier, quantitative differences in response were observed between these two classes of roots. The root tip of the primary root maintained an angle of 136 degrees to the horizontal as it traversed the barrier whereas the lateral roots adopted an angle of 154 degrees. Thus, this root tip angle appeared dependent on the gravitropic set-point angle of the root type with the difference in tracking angle quantitatively reflecting differences in initial set-point angle. Concave and convex barriers were also used to analyze the response of the root to tracking along a continuously varying surface. The roots maintained the a fairly fixed angle to gravity on the curved surface implying a constant resetting of this tip angle/tracking response as the curve of the surface changed. We propose that the interaction of touch and gravity sensing/response systems combine to strictly control the tropic growth of the root. Such signal integration is likely a critical part of growth control in the stimulus-rich environment of the soil.     

Seed-to-seed growth of Arabidopsis thaliana on the International Space Station.

The assembly of the International Space Station (ISS) as a permanent experimental outpost has provided the opportunity for quality plant research in space. To take advantage of this orbital laboratory, engineers and scientists at the Wisconsin Center for Space Automation and Robotics (WCSAR), University of Wisconsin-Madison, developed a plant growth facility capable of supporting plant growth in the microgravity environment. Utilizing this Advanced Astroculture (ADVASC) plant growth facility, an experiment was conducted with the objective to grow Arabidopsis thaliana plants from seed-to-seed on the ISS. Dry Arabidopsis seeds were anchored in the root tray of the ADVASC growth chamber. These seeds were successfully germinated from May 10 until the end of June 2001. Arabidopsis plants grew and completed a full life cycle in microgravity. This experiment demonstrated that ADVASC is capable of providing environment conditions suitable for plant growth and development in microgravity. The normal progression through the life cycle, as well as the postflight morphometric analyses, demonstrate that Arabidopsis thaliana does not require the presence of gravity for growth and development.     

Peculiarities of genital organ formation in Arabidopsis thaliana (L) Heynh. under spaceflight conditions.

An experiment was carried out ahoard the Salyut 6 research orbital station on Arabidopsis thaliana cultivations. The seeds were sprouted in the Svetoblok 1 device which provides for plant growth in the agar medium under sterile conditions and at 4000 lux illumination. The experimental plants, as well as the controls, reached approximately the same developmental stages: both flowered and began to bear fruit. A microscopic examination of the generative organs in the control and experimental plants shows that in normally formed (by appearance) flower buds and flowers of the experimental plants, as distinct from the controls, there were no fertile elements of the adroecium and gynoecium. Degeneration of the latter occurred at different stages of generative organ development. Possible reasons for this phenomenon in plants grown under weightless conditions are considered.     

Ultrastructural and functional changes of photosynthetic apparatus of Arabidopsis thaliana (L.) Heynh induced by clinorotation.

By the 28th day of growth upon a slowly rotating horizontal clinostat there had been a rearrangement of chloroplast organisation in the Arabidopsis thaliana mesophyll cells, changes in the native chlorophyll forms and alterations to the composition of the pigment-protein complex.     

Gravity and developmental plasticity.

Living organisms, especially plants, show some plasticity in their overall development, usually as a response to the external environment. Plasticity may apply not only to the external form of organisms but also to their physiology as well as to the detailed structure of their genome. A further example of plasticity may be developmental instability, where anomalous development seems to appear spontaneously, probably as a result of some transient environmental perturbation. Whether the absence of gravity would have sufficient impact on any living process to evoke a specific course of plastic development is unknown, though it is possible that in certain circumstances special forms, or 'agravimorphs', could be produced. Through such new forms, it should be possible to identify processes required for development in which 1 x g gravity is a necessary participant.     

Gravity sensing in moss protonemata.

Moss protonemata are a valuable system for studying gravitropism because both sensing and upward curvature (oriented tip growth) take place in the same cell. We review existing evidence, especially for Ceratodon purpureus, that addresses whether the mass that functions in sensing is that of amyloplasts that sediment. Recent experiments show that gravitropism can take place in media that are denser than the apical cell. This indicates that gravity sensing relies on an intracellular mass rather than that of the entire cell and provides further support for the starch-statolith hypothesis of sensing. Possible mechanisms for how amyloplast mass functions in sensing and transduction are discussed.     

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.     

Determination of the threshold acceleration for the gravitropic stimulation of cress roots and hypocotyls.

To determine the range of the threshold acceleration (a-threshold) for the gravitropic stimulation of Lepidium sativum L. roots and hypocotyls, experiments were performed on a centrifuge-clinostat with two-orthogonal axes. The rotation rate of the clinostat was 4 rpm (< or = 1.8 x 10(-4) g), while that of the centrifuge was from 3 to 17 rpm (3 x 10(-3) to 10(-1) g). The gravitropic response was determined: (i) after growth of roots and hypocotyls in their normal vertical position and subsequent gravitropic stimulation for 3 h by accelerations of 4 x 10(-3) to 10(-1) g, and (ii) after continuous stimulation in the lateral direction by centripetal accelerations of 4 x 10(-3) to 10(-1) g. The a-threshold was defined by an extrapolation of the regression line of R = p + rx, where x was either ln a or l/a for 3 h or a continuous stimulation, respectively. The a-threshold estimated after 3 h stimulation was equal to 2.6 x 10(-3) g for roots and 3.1 x 10(-3) g for hypocotyls. The threshold accelerations that were unable to evoke a gravitropic response even with continuous stimulation of cress roots and hypocotyls were approximately 3.1 x 10(-3) g and 3.6 x 10(-3) g, respectively. Increasing the stimulation acceleration up to 4.1 x 10(-3) g led to a statistically confirmed gravitropic response of a definite proportion of both the root and hypocotyl populations. In the experiments where acceleration and stimulation time were variable, the threshold dose (D-threshold) for roots was determined to be about 14 to 22 g x s, depending on the stimulation duration and the range of accelerations. The kinetics of gravitropic response at a near-threshold acceleration (4 x 10(-3) to 1.9 x 10(-2) g) differed from that at 1 g (horizontal stimulation). At low forces, the maximal response dependent on the magnitude of acceleration could not be enhanced by increasing the stimulation time up to at least 210 min.     

Experiments with air-dried seeds of Arabidopsis thaliana (L) Heynh. and Crepis capillaris (L) Wallr., aboard Salyut 6.

Space flight factors resulted in the accumulation of genetic damage in embryonic meristem cells of seeds of Arabidopsis thaliana and Crepis capillaris in flights of different duration (49, 226, 408 and 827 days) aboard the orbital station Salyut 6. As a result, the viability of seeds and seedlings was reduced, and the sterility of plants grown from seeds exposed on Salyut 6 was increased. The effect depended upon the flight duration. The data obtained suggest an acceleration of seed aging under flight conditions.     

Gravity and the cells of gravity receptors in mammals.

Two new findings, that crystals located in the inner ear gravity receptors of mammals have the internal organization requisite for the piezoelectric property, and that sensory hair cells of these same receptors possess contractile-appearing striated organelles, have prompted the author to model mammalian gravity receptors in the ear on the principles of piezoelectricity and bioenergetics. This model is presented and a brief discussion of its implications for the possible effects of weightlessness follows.     

Gravity and positional homeostasis of the cell.

Normally bilateralization takes place in the presence of the Earth's gravity which produces torque, shear, tension and compression acting upon the naked aggregates of cytoplasm in the zygote which is only stabilized by a weak cytoskeleton. In an initial examination of the effects of these quantities on development, an expression is derived to describe the tendency of torque to rotate the egg and reorganize its constituents. This expression yields the net torque resulting from buoyancy and gravity acting upon a dumbbell shaped cell with heavy and light masses at either end and "floating" in a medium. Using crude values for the variables, torques of 2.5 x l0(-13) to 8.5 x 10(-1) dyne-cm are found to act upon cells ranging from 6.4 micrometers to 31 mm (chicken egg). By way of camparison six microtubules can exert a torque of 5 x 10(-9) dyne-cm. (1) Gravity imparts torque to cells; (2) torque is reduced to zero as gravity approaches zero; and (3) torque is sensitive to cell size and particulate distribution. Cells must expend energy to maintain positional homeostasis against gravity. Although not previously recognized, Skylab 3 results support this hypothesis: tissue cultures used 58% more glucose on Earth than in space. The implications for developmental biology, physiology, genetics, and evolution are considered. At the cellular and tissue level the concept of "gravity receptors" may be unnecessary.     

Short-term and long-term clinostat and vibration-induced biochemical changes in dwarf marigold stems.

Stems of 21-day dwarf Marigold plants cultivated on the clinostat were compared with plants cultivated on vertical axis rotators ("vibrational controls") and stationary controls for long-term changes in cell wall composition. Stems of 21-day plants grown under stationary conditions and subsequently exposed to the clinostat for 24 hours were also analyzed. Among the long-tern markers, calciun, lignin, and protein-bound hemicellulose (possibly cell wall glycoprotein) clearly differentiated the effects of vibration from those of the clinostat. Short-term differential responses included rate of ethylene production, nastic movement and peroxidase activity of the cell wall, but not of the protoplast.     

Morphometric analysis of cell size patterning involved in gravitropic curvature of the stipe of Coprinus cinereus.

During gravitropic bending of the stipe of Coprinus cinereus the majority of elongation occurred in the apical region of the lower surface of the stipe, although some elongation was seen throughout the stipe. The final rate of elongation was similar at both the upper and lower stipe surfaces but the lower surface achieved this rate first (close to the reaction time 25 min), whilst the upper surface of the stipe only attained its final elongation rate after a period of acceleration of 150 min. Detailed morphometric analysis of cell size patterning in transverse sections revealed no significant differences in cross sectional area, spatial or proportional distribution of different cell types between the upper and lower regions of the gravitropic bend. Measurements of longitudinal cell size revealed significant differences in compartment size between the lower and upper region. Hyphal compartments of lower regions of the bend were on average four to five times longer than those of the upper region.     

Gravity and the membrane-solution interface: theoretical investigations.

The theory of concentration and potential variations at interfaces is applied to the membrane-solution interface to calculate density variations. The theory is modified to take care of the finite ion volumes in electrolytes. Our model is a phospholipid membrane with a surface charge density of -4.824*10(-6)(As/cm2) in contact with solutions of KCl, NaCl, CaCl2, and mixtures. Maximal density variations of about 4*10(-2)(G/cm3) were found in surface layers between the membrane and the solutions. The extension of the layers is in the range of 1 to 6 nm.     

Sedimentable amyloplasts in starch sheath cells of woody stems of Japanese cherry.

We examined whether sedimentable amyloplasts act as statolith in the perception of gravity in woody stems using the elongated internodes of Japanese cherry (Prunus jamasakura Sieb. ex Koidz.). In the internode of the seedlings grown on earth, amyloplasts were found sedimented at the distal end of each cell of the endodermal starch sheath tissue. In the internode grown on three-dimensional (3-D) clinostat, amyloplasts were dispersed throughout the cell matrix in the endodermal starch sheath tissue. After changing the positions of the internode from vertical to horizontal, re-sedimentation of amyloplasts toward the direction of gravity was completed in 1h, whereas the bending of the internode was observed after 12 days. We propose that sedimentable amyloplasts in the endodermal starch sheath cells may play a role in gravity perception leading to secondary xylem formation in the secondary thickening growth and eccentric growth in gravi-bending of tree stems.     

The gravitropic and phototropic responses of wheat grown in a space greenhouse prototype with hemispherical planting surface

The time course of gravicurvature of 3-day-old wheat (Triticum aestivum L., cv. Apogee) coleoptiles and 7-day-old wheat stems were studied in darkness and under red and red-blue light illumination after declination from the vertical at various angles. The experiments showed that the shortest gravitropic curvature corresponded to 30° initial angle of gravistimulation (IAG). The time course became longer as the IAG increased and with plant age. The effects of unilateral red (660 nm) and red-blue light (660 nm; 470 nm) at photosynthetic photon flux (PPF) of 30 μmol m⁻² s⁻¹ on the curvature of 3-day-old coleoptiles were evaluated. Red light did not produce phototropic bending of wheat coleoptiles in contrast with red-blue light. The analysis of experimental data showed that the curvature in response to a gravitropic stimulus or to combined gravity-light stimuli were not statistically different. Time course of gravitropic curvature were used to determine the acceptable crop rotation rate around the horizontal axis. Approximation of stem bending to a linear dynamic system described by a first-order aperiodic element with a lag allowed the determination of the dependence of the amplitude of apex oscillations on the rate of horizontal rotation under 1-g conditions. The calculated lowest minimal rotation rate (MRR) minimizing the gravitropic effects on wheat was about 1 revolution per hour (rph). Rotating the plant growth chamber (PGC) at a rate of more than MRR eliminated the effect of gravitropic curvature.     

Gravity, cellular membranes and associated processes: an introduction.

The aim of the session "Gravity: Cellular Membranes and Associated Processes" was to bring together scientists from different biological disciplines concentrated on the same scientific question: What are the basic interactions or influences, respectively, of gravity on cellular, molecular level? Presentations were selected dealing with the interaction of gravity with basic physico-chemical processes of membranes, such as changes of the membrane composition of human erythrocytes up to ultrastructural changes in a fungus and other objects after exposure to the conditions of space flight, clinorotation or increased acceleration by means of centrifugation. Taken together the data presented here and in the other session of the symposium on "Life and Gravity", clearly outline that future basic physico-chemical studies, and studies dealing with the molecular basis of the cellular signal-perception and transduction, have to be pressed forward in order to understand signal-responses on cellular level, but also of a whole organism.