Red light-induced suppression of gravitropism in moss protonemata.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.     

High temperature alters the growth reaction of Pottia protonemata.

Under gravistimulation, dark-grown protonemata of Pottia intermedia revealed negative gravitropism with a growth rate of approximately 28 μm·h⁻¹ at room temperature (20 °C). In 7 days, the protonema formed a bundle of vertically oriented filaments. At an elevated temperature (30 °C), bundles of vertically growing filaments were also formed. However, both filament growth rate and amplitude of the gravicurvature were reduced. Red light (RL) irradiation induced a positive phototropism of most apical protonemal cells at 20 °C. In a following period of darkness, approximately two-thirds of such cells began to grow upward again, recovering their negative gravitropism. RL irradiation at the elevated temperature caused a partial increase in the number of protonemal cells with negative phototropism, but the protonemata did not exhibit negative gravitropism after transfer to darkness. The negative gravitropic reaction was renewed only when protonemata were placed at 20 °C. A dramatic decrease in starch amount in protonemal apical cells, which are sensitive to both gravity and light, occurred at the higher temperature. Such a decrease may be one of the reasons for the inhibition of the protonemal gravireaction at the higher temperature. The observation has a bearing on the starch-statolith theory.     

Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots.

Phototropism as well as gravitropism plays a role in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism, but red light induces positive phototropism in Arabidopsis roots. Phytochrome A (phyA) and phyB mediate the positive red-light-based photoresponse in roots since single mutants (and the double phyAB mutant) were severely impaired in this response. In blue-light-based negative phototropism, phyA and phyAB (but not phyB) were inhibited in the response relative to the WT. In root gravitropism, phyB and phyAB (but not phyA) were inhibited in the response compared to the WT. The differences observed in tropistic responses were not due to growth limitations since the growth rates among all the mutants tested were not significantly different from that of the WT. Thus, our study shows that the blue-light and red-light systems interact in roots and that phytochrome plays a key role in plant development by integrating multiple environmental stimuli.     

High temperature alters the growth reaction of Pottia protonemata

Under gravistimulation, dark-grown protonemata of Pottia intermedia revealed negative gravitropism with a growth rate of approximately 28 μm·h⁻¹ at room temperature (20 °C). In 7 days, the protonema formed a bundle of vertically oriented filaments. At an elevated temperature (30 °C), bundles of vertically growing filaments were also formed. However, both filament growth rate and amplitude of the gravicurvature were reduced. Red light (RL) irradiation induced a positive phototropism of most apical protonemal cells at 20 °C. In a following period of darkness, approximately two-thirds of such cells began to grow upward again, recovering their negative gravitropism. RL irradiation at the elevated temperature caused a partial increase in the number of protonemal cells with negative phototropism, but the protonemata did not exhibit negative gravitropism after transfer to darkness. The negative gravitropic reaction was renewed only when protonemata were placed at 20 °C. A dramatic decrease in starch amount in protonemal apical cells, which are sensitive to both gravity and light, occurred at the higher temperature. Such a decrease may be one of the reasons for the inhibition of the protonemal gravireaction at the higher temperature. The observation has a bearing on the starch-statolith theory.     

Gravitropism and phototropism in protonemata of the moss Pohlia nutans (hedw.) Lindb

The gravitropism of protonemata of Pohlia nutans is described and compared with that of other mosses. In darkness, protonemata showed negative gravitropism. Under uniform illumination they grew radially over the substrate surface, whereas unilateral illumination induced positive phototropic growth. Gravitropism was coupled with starch synthesis and amyloplast formation. Protonematal gravitropic growth is more variable than the strict negative gravitropism of Ceratodon chloronema.     

Gravitropism and phototropism in protonemata of the moss Pohlia nutans (Hedw.) Lindb.

The gravitropism of protonemata of Pohlia nutans is described and compared with that of other mosses. In darkness, protonemata showed negative gravitropism. Under uniform illumination they grew radially over the substrate surface, whereas unilateral illumination induced positive phototropic growth. Gravitropism was coupled with starch synthesis and amyloplast formation. Protonematal gravitropic growth is more variable than the strict negative gravitropism of Ceratodon chloronema.     

Gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.

Shoots of higher plants exhibit negative gravitropism. However, little is known about the site of gravity perception in shoots and the molecular mechanisms of shoot gravitropic responses. Our recent analysis using shoot gravitropism 1(sgr1)/scarecrow(scr) and sgr7/short-root (shr) mutants in Arabidopsis thaliana indicated that the endodermis is essential for shoot gravitropism and strongly suggested that the endodermis functions as the gravity-sensing cell layer in dicotyledonous plant shoots. In this paper, we present our recent analysis and model of gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.     

Effects of a hypogeomagnetic field on gravitropism and germination in soybean

Any plants grown during long-term space missions will inevitably experience an extremely low magnetic field (i.e. a hypogeomagnetic field, HGMF). It is possible that the innate adaptation of plants to the earth’s magnetic field (i.e. the geomagnetic field, GMF) would be disrupted. Effects of the HGMF on plant physiological and metabolic processes are unclear. In this study we established a hypogeomagnetic incubation system on the ground and investigated the effects of the HGMF on the gravitropism and germination of soybean seeds. The gravitropism angle, germination percentage, germination speed, water absorbance ratio, seed weight, radicle length, radicle weight, and radicle weight ratio of soybean seeds grown in the local field and the HGMF were compared. In general, the gravitropism angle in the HGMF was smaller than that in the local field when seeds were positioned before emergence in such a way that the direction of the radicle was opposite to that of gravity. The germination percentage, germination speed, and radicle weight ratio increased in the HGMF compared to the control. Our results indicate that the germination and gravitropism of soybean seeds are affected by elimination of the geomagnetic field.     

The role of calcium in the regulation of hormone transport in gravistimulated roots.

Prior research has shown that gravistimulation induces preferential movement of calcium toward the lower side of the tips of maize roots and that roots depleted of calcium show impaired gravitropism. To further investigate the role of calcium in root gravitropism, we examined the effects of calcium on auxin movement in both vertical and gravistimulated roots of maize. Longitudinal movement of auxin was basipetally polar in intact roots but acropetally polar in decapped roots. Treatment of the root tip with calcium increased basipetal auxin movement in both intact and decapped roots. Gravistimulation induced asymmetric auxin movement toward the lower side of the root tip. Both asymmetric auxin movement and gravicurvature were inhibited by treatment of the root tip with auxin transport inhibitors or with EGTA. The results indicate that there is a close correlation between curvature and gravity-induced asymmetric auxin movement across the root cap. Since gravistimulation causes calcium movement toward the lower side of the root tip, our observation that calcium promotes basipetal auxin movement supports the idea that gravity-induced calcium asymmetry is a key step linking gravistimulation to the establishment of auxin asymmetry during root gravitropism.     

Gravity-dependent reactions of the moss Pohlia nutans protonemata.

In darkness, protonemata of Pohlia nutans (Hedw.) grew negatively gravitropically (upwards). However, not all filaments became gravitropic immediately after transfer to darkness. Some of them (~20%) for several days grew in different directions with respect to gravity. The apical cells of those protonemata predominantly contained multiple chloroplasts. The intensity of chlorophyll fluorescence rapidly decreased in the apical cells of such protonemata while starch content increased in comparison with upright growing protonemata. Light, especially in the red and blue part of the spectrum, inhibited protonemal gravitropism. Red light induced stronger inhibitory effects than blue light. Red light of 1.0 to 1.5 micromoles m-2 s-1 intensity induced bud differentiation in apical cells on almost all side branches of main protonemal filaments. Bright fluorescence of F-actin bundles in the tip of apical protonematal cells and a delicately fluorescing network enclosing plastids basal to the tip in a sedimentation zone were visualized. Bright fluorescence of actin as local patches and fine prominent axially oriented bundles was observed in cells of gametophore buds.     

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.     

What is the threshold amount of starch necessary for full gravitropic sensitivity?

In preparation for microgravity experiments, we studied the kinetics of gravitropism in seedlings of wild-type (WT) Arabidopsis and three starch-deficient mutants. One of these mutants is starchless (ACG 21) while the other two are intermediate starch mutants (ACG 20 and ACG 27). In root cap cells, ACG 20 and 27 have 51% and 60% of the WT amount of starch, respectively. However, in endodermal cells of the hypocotyl, ACG 20 has a greater amount of starch than ACG 27. WT roots and hypocotyls were much more responsive to gravity than were the respective organs of the starchless mutant, and the intermediate starch mutants exhibited reduced gravitropism but had responses that were close to that of the WT. In roots, ACG 27 (more starch) was more responsive than ACG 20 (less starch), while in hypocotyls, ACG 20 (more starch) had a greater response than ACG 27 (less starch). Taken together, our 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. These results also suggest that (in roots) 51-60% starch is close to the threshold amount of starch needed for full gravitropism and that the gravity sensing system is "overbuilt."     

Cell biology of plant gravity sensing.

The debate about whether gravity sensing relies upon statoliths (amyloplasts that sediment) has intensified with recent findings of gravitropism in starchless mutants and of claims of hydrostatic gravity sensing. Starch and significant plastid sedimentation are not necessary for reduced sensing in mutant roots, but plastids might function here if there were a specialized receptor for plastid mass e.g. in the ER. Alternatively, components in addition to amyloplasts might provide mass for sensing. The nucleus is dense and its position is regulated, but no direct data exist for its role in sensing. If the weight of the protoplast functioned in sensing, why would there be specific cytological specializations favoring sedimentation rather than cell mass? Gravity has multiple effects on plants in addition to gravitropism. There may be more than one mechanism of gravity sensing.     

Microinjection--a tool to study gravitropism.

Despite extensive studies on plant gravitropism this phenomenon is still poorly understood. The separation of gravity sensing, signal transduction and response is a common concept but especially the mechanism of gravisensing remains unclear. This paper focuses on microinjection as powerful tool to investigate gravisensing in plants. We describe the microinjection of magnetic beads in rhizoids of the green alga Chara and related subsequent manipulation of the gravisensing system. After injection, an external magnet can control the movement of the magnetic beads. We demonstrate successful injection of magnetic beads into rhizoids and describe a multitude of experiments that can be carried out to investigate gravitropism in Chara rhizoids. In addition to examining mechanical properties, bead microinjection is also useful for probing the function of the cytoskeleton by coating beads with drugs that interfere with the cytoskeleton. The injection of fluorescently labeled beads or probes may reveal the involvement of the cytoskeleton during gravistimulation and response in living cells.     

Comparing plant and fungal gravitropism using imitational models based on reiterative computation.

Mathematical models which imitate plant gravitropic responses were used to compare plant and fungal gravitropism with kinetic data from the agarics Coprinus cinereus and Flammulina velutipes. Similarities were: bending depends on differential growth; growth of the organ is most intensive just behind the apex; gravitropisms exhibit a substantial time delay. Differences were: the agaric stem apex always returns to the vertical (some plant organs show stable plagiogravitropic growth); curvature compensation occurred in C. cinereus; C. cinereus stems rarely overshot or oscillated around the vertical although data for F. velutipes showed a single overshoot and oscillation. The work focused attention on the need for data on detection-level thresholds, angle-response and acceleration-response relationships in fungi, and the need for detailed observations of gravitropism kinetics in a larger number and wider range of fungi.     

Physiological characterization of gravitaxis in Euglena gracilis and Astasia longa studied on sounding rocket flights.

Euglena gracilis is a photosynthetic, unicellular flagellate found in eutrophic freshwater habitats. The organisms control their vertical position in the water column using gravi- and phototaxis. Recent experiments demonstrated that negative gravitaxis cannot be explained by passive buoyancy but by an active physiological mechanism. During space experiments, the threshold of gravitaxis was determined to be between 0.08 and 0.12 x g. A strong correlation between the applied acceleration and the intracellular cAMP and Ca2+ was observed. The results support the hypothesis, that the cell body of Euglena, which is denser than the surrounding medium exerts a pressure onto the lower membrane and activates mechanosensitive Ca2+ channels. Changes in the membrane potential and the cAMP concentration are most likely subsequent elements in a signal transduction chain, which results in reorientation strokes of the flagellum.     

The gravireaction of Ceratodon protonemata treated with gibberellic acid.

Moss protonemata exhibit negative gravitropism and the amyloplasts of the apical cell seem to play a key role in protonemal gravisensitivity. However, the mechanisms of this process are still poorly understood. Previously, we have shown that Ceratodon protonemata grown on agar-medium demonstrated greater gravicurvature than protonemata grown on medium with 11 mM glucose. In this study, we have examined whether gibberellic acid (GA), which promotes alpha-amylase expression, influences graviresponse of C. purpureus protonemata (strains WT-4 and WT-U) and how this event interacts with exogenous soluble sugars. After gravistimulation the WT-4 strain curved about twice as fast as the WT-U strain. However, responses of both strains to added substances were similar. High concentration of glucose (0.11 M) caused a decrease in protonema curvature, while the same concentration of sucrose did not significantly change the angles of curvature compared with controls. GA at 0.1 mM and higher concentrations inhibited gravitropism, and caused some apical cells to swell. The possible involvement of the carbohydrates in gravitropism is discussed.     

The first dedicated Life Sciences mission--Spacelab 4.

Spacelab is a large versatile laboratory carried in the bay of the Shuttle Orbiter. The first Spacelab mission dedicated entirely to Life Sciences is known as Spacelab 4. It is scheduled for launch in late 1985 and will remain aloft for seven days. This payload consists of 25 tentatively selected investigations combined into a comprehensive integrated exploration of the effects of acute weightlessness on living systems. An emphasis is placed on studying physiological changes that have been previously observed in manned space flight. This payload has complementary designs in the human and animal investigations in order to validate animal models of human physiology in weightlessness. The experimental subjects include humans, squirrel monkeys, laboratory rats, several species of plants, and frog eggs. The primary scientific objectives include study of the acute cephalic fluid shift, cardiovascular adaptation to weightlessness, including postflight reductions in orthostatic tolerance and exercise capacity, and changes in vestibular function, including space motion sickness, associated with weightlessness. Secondary scientific objectives include the study of red cell mass reduction, negative nitrogen balance, altered calcium metabolism, suppressed in vitro lymphocyte reactivity, gravitropism and photropism in plants, and fertilization and early development in frog eggs. The rationale behind this payload, the selection process, and details of the individual investigations are presented in this paper.     

The promotive effect of latrunculin B on maize root gravitropism is concentration dependent.

The cytoskeleton has been proposed to be a key player in the gravitropic response of higher plants. A major approach to determine the role of the cytoskeleton in gravitropism has been to use inhibitors to disrupt the cytoskeleton and then to observe the effect that such disruption has on organ bending. Several investigators have reported that actin or microtubule inhibitors do not prevent root gravitropism, leading to the conclusion that the cytoskeleton is not involved in this process. However, there are recent reports showing that disruption of the actin cytoskeleton with the actin inhibitor, latrunculin B, promotes the gravitropic response of both roots and shoots. In roots, curvature is sustained during prolonged periods of clinorotation despite short periods of gravistimulation. These results indicate that an early gravity-induced signal continues to persist despite withdrawal of the constant gravity stimulus. To investigate further the mechanisms underlying the promotive effect of actin disruption on root gravitropism, we treated maize roots with varying concentrations of latrunculin B in order to determine the lowest concentration of latrunculin B that has an effect on root bending. After a 10-minute gravistimulus, treated roots were axially rotated on a one rpm clinostat and curvature was measured after 15 hours. Our results show that 100 nM latrunculin B induced the strongest promotive effect on the curvature of maize roots grown on a clinostat. Moreover, continuously gravistimulated roots treated with 100 nM latrunculin B exhibited stronger curvature responses while decapped roots treated with this concentration of latrunculin B did not bend during continuous gravistimulation. The stronger promotive effect of low concentrations of latrunculin B on the curvature of both clinorotated and continuously gravistimulated roots suggests that disruption of the finer, more dynamic component of the actin cytoskeleton could be the cause of the enhanced tropic responses of roots to gravity.