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.     

Gravity effects on the [correction of thr] growth and development of moss secondary protonemata.

The superficial cells of dark-grown moss shoots give rise to negatively gravitropic protonemata, whatever the orientation of the shoot. Shoot orientation, however, does affect from which side of the shoot the protonemata form and the direction of their growth. Protonemata from horizontal shoots grow out at a near-right angle to their supporting axes and are initiated more or less evenly along the upper side of the stem. Protonemata arising from vertically-oriented shoots in either an upright or an inverted position grow straight at an acute angle to the stem axis. The difference in the growth direction of the protonemata seems to be conditioned by the different position of the growth zone of the protonemal outgrowths, and subsequently that of the apical protonemal cells, with respect to the gravity vector. Observations suggest that the shoot protonemata, in conditions of clinorotation, persist in their original growth direction. Results also indicate that, in darkness, gravity determines only the site of protonemata initiation, not the process of initiation itself. Light, by contrast, by acting through both phytochrome and high-energy reaction systems, triggers the initiation process and defines the location of protonemata.     

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.     

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.     

Influence of gravity on spatial orientation and morphogenesis of moss sporophytes.

During the growth and development of the sporophytic capsules of some moss species, negative gravitropism is changed for a positive one. Horizontal clinostat rotation induced unregulated growth of the sporophytes and their twisting; some of sporophytes remained straight, however. It has been established that the change of the gravitropic reaction is related to capsule formation and to the redistribution of amyloplast cells of the sporophyte graviperception zone.     

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.     

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.     

Influence of gravity on the circadian timing system.

The circadian timing system (CTS) is responsible for daily temporal coordination of physiological and behavioral functions both internally and with the external environment. Experiments in altered gravitational environments have revealed changes in circadian rhythms of species ranging from fungi to primates. The altered gravitational environments examined included both the microgravity environment of spaceflight and hyperdynamic environments produced by centrifugation. Acute exposure to altered gravitational environments changed homeostatic parameters such as body temperature. These changes were time of day dependent. Exposure to gravitational alterations of relatively short duration produced changes in both the homeostatic level and the amplitude of circadian rhythms. Chronic exposure to a non-earth level of gravity resulted in changes in the period of the expressed rhythms as well as in the phase relationships between the rhythms and between the rhythms and the external environment. In addition, alterations in gravity appeared to act as a time cue for the CTS. Altered gravity also affected the sensitivity of the pacemaker to other aspects of the environment (i.e., light) and to shifts of time cues. Taken together, these studies lead to the conclusion that the CTS is indeed sensitive to gravity and its alterations. This finding has implications for both basic biology and space medicine.     

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.     

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.     

Influence of zero gravity simulation on time course of mitosis in microplasmodia of Physarum polycephalum.

Detrimental effects of weigntlessness are no longer expected to hinder successful mitosis. Experiments in space and on the fast clinostat give no hints of this. Nevertheless we are thinking of a g sensitivity during the process of chromosome condensation and distribution. The time course of nuclear division in microplasmodia of the slime mold Physarum polycephalum was investigated under 0 g simulation on the fast rotating clinostat in comparison to 1 g controls. The result of this experiment is: A significant shortening of mitosis under 0 g simulation compared to 1 g controls.     

Possible actions of gravity on the cellular machinery.

Since the first flight of the ESA Biorack on the German Spacelab Mission D1 in 1985 evidence has been obtained that biological cells and small unicellular organisms function differently under conditions of microgravity. However, there is still lack of scientific proof that these effects are caused by a direct influence on the cells in the weightlessness condition. The question how normal gravity may play a role in cellular activity is being addressed and the results show that gravity may provide important signals during certain state transitions in the cell. These would be gravity-sensitive windows in the biological process. Also, by amplification mechanisms inside the cell, the cell may assume a state that is typical for normal gravity conditions and would change in microgravity. Experimental tools are discussed that would provide the conditions to obtain evidence for direct action of gravity and for the possible existence of gravity-sensitive windows.     

Influence of altered gravity on brain cellular energy and plasma membrane metabolism of developing lower aquatic vertebrates.

Biochemical analyses of the brain of cichlid fish larvae, exposed for 7 days to increased acceleration of 3g (hyper-g), revealed an increase in energy availability (succinate dehydrogenase activity, SDH), and in mitochondrial energy transformation (creatine kinase, Mia-CK), but no changes in an energy consumptive process (high-affinity Ca(2+)-ATPase). Brain glucose-6-phosphate dehydrogenase (G6PDH) of developing fish was previously found to be increased after hyper-g exposure. Three respectively 5 hours thereafter dramatic fluctuations in enzyme activity were registered. Analysing the cytosolic or plasma membrane-located brain creatine kinase (BB-CK) of clawed toad larvae after long-term hyper-g exposure a significant increase in enzyme activity was demonstrated, whereas the activity of a high affinity Ca(2+)-ATPase remained unaffected.     

Effect of gravity on the velocity of a crystallization front

The crystal growth from a liquid can be studied by methods of non-equilibrium thermodynamics of a discontinuity surface. These methods are used to derive the dependence of the crystalization velocity on transport and surface phenomena across and along an interface solid-liquid and on the acceleration of gravity.     

Effects of gravity on early development.

The development of embryonic and larval stages of the South African Toad Xenopus laevis D, was investigated in hyper-g up to 5 g (centrifuge), in simulated 0 g (fast-rotating clinostat), in alternating low g, hyper-g (parabolic flights) and in microgravity (Spacelab missions D1, D-2). The selected developmental stages are assumed to be very sensitive to environmental stimuli. The results showed that the developmental reaction processes run normal also in environments different to 1 g and that aberrations in behavior and morphology normalize after return to 1 g. Development, differentiation, and morphology of the gravity perceiving parts of the vestibular system (macula-organs) had not been affected by exposure to different g-levels.     

Effects of altered gravity on the swimming behaviour of fish.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness-phenomena (SMS, a kinetosis). It has been argued that SMS during PAFs might not be based on microgravity alone but rather on changing accelerations from 0 g to 2 g. We test here the hypothesis that PAF-induced kinetosis is based on asymmetric statoliths (i.e., differently weighed statoliths on the right and the left side of the head), with asymmetric inputs to the brain being disclosed at microgravity. Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated (1) whether or not kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry and (2) whether or not slow translational and continuously changing linear (vertical) acceleration on ground induced kinetosis. These latter accelerations were applied using a specially developed parabel-animal-container (PAC) to stimulate the cupular organs. The results suggest that the fish tested on ground can counter changing accelerations successfully without revealing kinetotic swimming patterns. Kinetosis could only be induced by PAFs. This finding suggests that it is indeed microgravity rather than changing accelerations, which induces kinetosis. Moreover, we demonstrate that fish swimming kinetotically during PAFs correlates with a higher otolith asymmetry in comparison to normally behaving animals in PAFs.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

The influence of gravity on the process of development of animal systems

The development of animal systems is described in terms of a series of overlapping phases: pattern specification; differentiation; growth; and aging. The extent to which altered (micro) gravity (g) affects those phases is briefly reviewed for several animal systems. As a model, amphibian egg/early embryo is described. Recent data derived from clinostat protocols indicates that microgravity simulation alters early pattern specification (dorsal/ventral polarity) but does not adversely influence subsequent morphogenesis. Possible explanations for the absence of catastrophic microgravity effects on amphibian embryogenesis are discussed.