Gravitropism of basidiomycetous fungi--on Earth and in microgravity.

In order to achieve perfect positioning of their lamellae for spore dispersal, fruiting bodies of higher fungi rely on the omnipresent force gravity. Only accurate negatively gravitropic orientation of the fruiting body cap will guarantee successful reproduction. A spaceflight experiment during the STS-55 Spacelab mission in 1993 confirmed that the factor gravity is employed for spatial orientation. Most likely every hypha in the transition zone between the stipe and the cap region is capable of sensing gravity. Sensing presumably involves slight sedimentation of nuclei which subsequently causes deformation of the net-like arrangement of F-actin filament strands. Hyphal elongation is probably driven by hormone-controlled activation and redistribution of vesicle traffic and vesicle incorporation into the vacuoles and cell walls to subsequently cause increased water uptake and turgor pressure. Stipe bending is achieved by way of differential growth of the flanks of the upper-most stipe region. After reorientation to a horizontal position, elongation of the upper flank hyphae decreases 40% while elongation of the lower flank slightly increases. On the cellular level gravity-stimulated vesicle accumulation was observed in hyphae of the lower flank.     

Gravisensing in single-celled systems: characean rhizoids and protonemata.

Gravitropically tip-growing cell types are attractive unicellular model systems for investigating the mechanisms and the regulation of gravitropism. Especially useful for studying the mechanisms of positive and negative gravitropic tip-growth are characean rhizoids and protonemata. They originate from the same cell type, show the same overall cell shape, cytoplasmic zonation, arrangement of actin and microtubule cytoskeleton, use statoliths for gravisensing, but show opposite gravitropism. In both cell types, actin microfilaments are complexly organized in the apical dome,where a dense spherical actin array is colocalized with spectrin-like epitopes and a unique endoplasmic reticulum aggregate, the structural center of the Spitzenk?rper. The opposite gravitropic responses seem to be based on differences in the actin-organized anchorage of the Spitzenk?rper and the actin-mediated transport of statoliths. In negatively gravitropic (upward bending) protonemata, the statoliths-induced drastic upward shift of the cell tip is preceded by a relocalization of dihydropyridine-binding calcium channels and of the apical calcium gradient to the upper flank (bending by bulging). Such relocalizations have not been observed in positively gravitropically responding (downward growing) rhizoids in which statoliths sedimentation is followed by differential flank growth (bending by bowing). This paper reviews the current knowledge and hypotheses on the mechanisms of the opposite gravitropic responses in characean rhizoids and protonemata.     

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.     

Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.     

Gravisensing: ionic responses, cytoskeleton and amyloplast behavior.

In Zea mays L., changes in orientation of stems are perceived by the pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. Gravity is perceived in the bundle sheath cells, which contain amyloplasts that sediment to the new cell base when a change in the gravity vector occurs. The mechanism by which the mechanical signal is transduced into a physiological response is so far unknown for any gravity perceiving tissue. It is hypothesized that this involves interactions of amyloplasts with the plasma membrane and/or ER via cytoskeletal elements. To gain further insights into this process we monitored amyloplast movements in response to gravistimulation. In a pharmacological approach we investigated how the dynamics of plastid sedimentation are affected by actin and microtubule (MT) disrupting drugs. Dark grown caulonemal filaments of the moss Physcomitrella patens respond to gravity vector changes with a reorientation of tip growth away from the gravity vector. MT distributions in tip cells were monitored over time and MTs were seen to accumulate preferentially on the lower flank of the tip 30 min after a 90 degree turn. Using a self-referencing Ca2+ selective ion probe, we found that growing caulonemal filaments exhibit a Ca2+ influx at the apical dome, similar to that reported previously for other tip growing cells. However, in gravistimulated Physcomitrella filaments the region of Ca2+ influx is not confined to the apex, but extends about 60 micrometers along the upper side of the filament. Our results indicate an asymmetry in the Ca2+ flux pattern between the upper and side of the filament suggesting differential activation of Ca2+ permeable channels at the plasma membrane.     

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.     

An introduction to gravity perception in plants and fungi--a multiplicity of mechanisms.

The origin and subsequent evolution of life on Earth has taken place within an environment of which a 1g gravitational force is a part. Thus, all living organisms accommodate this variable in their structure and function. Evolution has also selected mechanisms to sense gravity which, in consequence, give particular orientations to living process. It is anticipated that the higher the evolutionary status of an organism, the greater the chance that it will possess multiple mechanisms of gravisensing because evolution discards nothing that assists fitness, but only adds to existing processes. A multiplicity of mechanisms permits gravity to participate in a wide range of developmental programmes, such as taxes, morphisms and tropisms, through the action of different sensors and distinct transduction/response pathways.     

Laser ablation of root cap cells: implications for models of graviperception.

The initial event of gravity perception by plants is generally thought to occur through sedimentation of amyloplasts in specialized sensory cells. In the root, these cells are the columella which are located toward the center of the root cap. To define more precisely the contribution of columella cells to root gravitropism, we used laser ablation to remove single columella cells or groups of these cells and observed the effect of their removal on gravity sensing and response. Complete removal of the cap or all the columella cells (leaving peripheral cap cells intact) abolishes the gravity response of the root. Removal of stories of columella revealed differences between regions of the columella with respect to gravity sensing (presentation time) versus graviresponse (final tropic growth response of the root). This fine mapping revealed that ablating the central columella located in story 2 had the greatest effect on presentation time whereas ablating columella cells in story 3 had a smaller or no effect. However, when removed by ablation the columella cells in story 3 did inhibit gravitropic bending, suggesting an effect on translocation of the gravitropic signal from the cap rather than initial gravity perception. Mapping the in vivo statolith sedimentation rates in these cells revealed that the amyloplasts of the central columella cells sedimented more rapidly than those on the flanks do. These results show that cells with the most freely mobile amyloplasts generate the largest gravisensing signal consistent with the starch statolith hypothesis of gravity sensing in roots.     

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.     

Intracellular magnetophoresis of statoliths in Chara rhizoids and analysis of cytoplasm viscoelasticity.

The statoliths in Chara rhizoids are denser and more diamagnetic than the cytoplasm, therefore they can be displaced inside a living cell by a sufficiently strong high gradient magnetic field (HGMF). An experimental setup for intracellular magnetophoresis of statoliths was developed. The movement of statoliths and rhizoid growth was measured by video microscopy either under the influence of gravity or a HGMF equivalent to about 2 g. The contribution of the cytoskeleton to statolith motility was assayed before and after depolymerizing microtubules with oryzalin and F-actin with latrunculin B. Application of latrunculin caused immediate cessation of growth, clumping of statoliths, and application of HGMF resulted in higher displacement of statoliths. Oryzalin had no effect on the behavior of statoliths. The data indicate that magnetophoresis is a useful tool to study the gravisensing system and rheology of the Chara rhizoid.     

Gravimorphogenesis and ultrastructure of the fungus Flammulina velutipes grown in space, on clinostats and under hyper-g conditions.

The D-2-mission provided the facilities to cultivate the higher basidomycete Flammulina velutipes (Agaricales) in space for about 8 days. Gravimorphogenesis of developing fruiting body primordia in weightlessness was documented in comparison to cultures incubated on a 1xg reference centrifuge in space. Chemical fixation of fruiting bodies took place for later ultrastructural analysis. The microgravity grown fruiting bodies exhibited random orientation compared to the 1xg-cultures where fruiting bodies showed exactly negative gravitropic orientation. Weightlessness did not impair fruiting body morphogenesis and growth although flat and helically twisted stipes were observed. Ultrastructural analyses of microgravity-, 1xg- and 20xg-samples did not reveal sedimentable cell components. Gravitropic bending involves growth inhibition at the upper side of a horizontally oriented transition zone, the graviperceptive region of the stipe. The fastest ultrastructural response to the altered direction of the accelerational force is the accumulation of cytosolic vesicles at the lower part of this region. They contribute to the expansion of the central vacuole and therefore to the differential enlargement of the lower side of the stipe.     

Gravisensing in plants and fungi.

The principle of establishing and maintaining a gravitropic set point angle depends on gravisensing and a subsequent cascade of events that result in differential elongation of the responsive structures. Since gravity acts upon masses, the gravisensing mechanisms of all biological systems must follow the same principle, namely the sensing of some force due to differential acceleration of the perceiving entity and a reference structure. This presentation will demonstrate that gravisensing can be accomplished by various means, ranging from cytoskeletal organization, mechano-elastic stress to perturbation of electric signals. However, several arguments indicate that sedimentation of either dense plastids (statoliths), the entire protoplast, or a combination of these represents the primary step in graviperception in plants. In fungi, nuclei and cytoskeletal proteins are believed to form a network capable of gravisensing but sedimenting organelles that may function as statoliths have been identified. Theoretical and practical limitations of gravisensing and detection of acceleration forces necessitate microgravity experiments to identify the primary perceptor, subsequent biochemical mechano-transduction, and biological response processes.     

Ultrastructure and cytoskeleton of Chara rhizoids in microgravity.

Gravitropic tip growth of Chara rhizoids is dependent on the presence and functional interaction between statoliths, cytoskeleton and the tip-growth-organizing complex, the Spitzenkorper. Microtubules are essential for the polar cytoplasmic zonation but are excluded from the apex and do not play a crucial role in the primary steps of gravisensing and graviresponse. Actin filaments form a dense meshwork in the subapical zone and converge into a prominent apical actin patch which is associated with the endoplasmic reticulum (ER) aggregate representing the structural center of the Spitzenkorper. The position of the statoliths is regulated by gravity and a counteracting force mediated by actomyosin. Reducing the acceleration forces in microgravity experiments causes a basipetal displacement of the statoliths. Rhizoids grow randomly in all directions. However, they express the same cell shape and cytoplasmic zonation as ground controls. The ultrastructure of the Spitzenkorper, including the aggregation of ER, the assembly of vesicles in the apex, the polar distribution of proplastids, mitochondria, dictyosomes and ER cisternae in the subapical zone is maintained. The unaltered cytoskeletal organization, growth rates and gravitropic responsiveness indicate that microgravity has no major effect on gravitropic tip-growing Chara rhizoids. However, the threshold value of gravisensitivity might be different from ground controls due to the altered position of statoliths, a possibly reduced amount of BaSO4 in statoliths and a possible adaptation of the actin cytoskeleton to microgravity conditions.     

Theoretical considerations of plant gravisensing.

The mechanisms proposed to explain gravity sensing can be divided into two groups, "statolith" and "non-statolith" mechanisms. The traditional estimates of the plausibility of these mechanisms are based on the analysis of the signal-to-noise ratio. The existing data indicate that the problem of plant gravisensing may be related to the general problem of the detection of weak signals in mechanoreceptors. This paper reviews the known mechanisms of plant gravisensing as well as the latest nonlinear stochastic models of mechanoreception in which noise promotes detection and amplification of weak signals. These models based on nonlinear stochastic phenomena may be used to explain plant gravisensing, if the cell is considered a dynamic, spatially distributed system of active intracellular cytoskeletal networks and mechanosensitive proteins.     

Gravitropism and phototropism of oat coleoptiles: post-tropic autostraightening and tissue shrinkage during tropism.

We measured changes in length on the two opposite sides of the red-light-grown oat (Avena sativa L.) coleoptiles subjected to either gravitropic or phototropic stimulation and subsequently rotated on a horizontal clinostat. The length measurement was conducted using three 5 mm-long zones delimited by ink markers from the tip. Curvature of each zone was analyzed from the length difference between the two sides. Gravitropism was induced by displacing the seedling from the vertical by 30 degrees or 90 degrees for 25 min. Phototropism was induced by exposing the coleoptile to unilateral blue light for 30 s, which provided a fluence (1.0 micromoles m-2) optimal for the pulse-induced positive phototropism or a lower, suboptimal fluence (0.03 micromoles m-2). After negatively gravitropic bending, the upper two zones straightened rapidly at either displacement angle. After positively phototropic bending, straightening occurred, but only in the top zone and at the lower fluence. The upper two zones straightened rapidly, however, when bilateral blue light (30 s; 15 micromoles m-2 from either direction) was applied 25 min after unilateral stimulation at the higher fluence. Bilateral blue light alone induced no curvature. These results confirm that the straightening of gravitropically bent coleoptiles is autonomic, and suggest that a similar autonomic response participates in the straightening of phototropically bent coleoptiles. Suppression of elongation on the concave side of the coleoptile mainly accounted for gravitropic and phototropic curvatures. The concave side of the top zone shrank during both tropisms. This shrinkage progressed at a high rate from the beginning of curvature response, suggesting that a drop in turgor pressure is the main and direct cause of the shrinkage.     

Characterization of a novel gravitropic mutant of morning glory, weeping2

In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 (we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT (PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.     

Graviresponses in fungi.

Although the orientation of mycelial hyphal growth is usually independent of the gravity vector, individual specialised hyphae can show response to gravity. This is exemplified by the sporangiophore of Phycomyces, but the most striking gravitropic reactions occur in mushroom fruit bodies. During the course of development of a mushroom different tropisms predominate at different times; the young fruit body primordium is positively phototropic, but negative gravitropism later predominates. The switch between tropisms has been associated with meiosis. The spore-bearing tissue is positively gravitropic and responds independently of the stem. Bracket polypores do not show tropisms but exhibit gravimorphogenetic responses: disturbance leads to renewal of growth producing an entirely new fruiting structure. Indications from both clinostat and space flown experiments are that the basic form of the mushroom (overall tissue arrangement of stem, cap, gills, hymenium, veil) is established independently of the gravity vector although maturation, and especially commitment to the meiosis-sporulation pathway, requires the normal gravity vector. The gravity perception mechanism is difficult to identify. The latest results suggest that disturbance of cytoskeletal microfilaments is involved in perception (with nuclei possibly being used as statoliths), and Ca2(+)-mediated signal transduction may be involved in directing growth differentials.     

激光弯曲机理的试验研究

通过对试件在激光与材料非熔凝作用下动态变形过程的实时测量及光斑中心处横截面的金相分析,得知激光弯曲变形是热应变和相变共同作用的结果.试验结果表明:激光弯曲变形的大小和方向取决于材料吸收的能量、材料特性和材料的厚度,并给出了这些影响因素的变化规律.研究结果进一步完善了激光弯曲的变形机理,并为进一步研究激光弯曲过程奠定了基础.     

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.     

Growth of Prunus tree stems under simulated microgravity conditions.

Stem growth of Prunus trees under simulated microgravity conditions was examined using a three-dimensional clinostat. The stems elongated with bending under such conditions. Stem elongation and leaf expansion were both promoted, whereas the formation of xylem in the secondary thickening growth was inhibited under the simulated microgravity condition. In secondary xylem, sedimentable amyloplasts were observed in the 1g control. The present results suggest that stem elongation and leaf expansion may be inhibited at 1g, while growth direction and secondary xylem formation depend on a gravity stimulus. A space experiment is expected to advance research on thickening growth in trees.