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.     

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.     

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-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.     

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.     

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.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

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.     

Statolith positioning by microfilaments in Chara rhizoids and protonemata.

The rhizoids of the green alga Chara are tip-growing cells with a precise positive gravitropism. In rhizoids growing downwards the statoliths never sediment upon the cell wall at the very tip but keep a minimal distance of approximately 10 micrometers from the cell vertex. It has been argued that this position is attained by a force acting upon the statoliths in the basal direction and that this force is generated by an interaction between actin microfilaments and myosin on the statolith membrane. This hypothesis received experimental support from (1) effects of the actin-attacking drug cytochalasin, (2) experiments under microgravity conditions, and (3) clinostat experiments. Using video-microscopy it is now shown that this basipetal force also acts on statoliths during sedimentation. As a result, many statoliths in Chara rhizoids do not simply fall along the plumb line while sedimenting during gravistimulation, but move basipetally. This statolith movement is compared to the ones occurring in the unicellular Chara protonemata during gravistimulation. Dark-grown protonemata morphologically closely resemble the rhizoids but respond negatively gravitropic. In contrast to the rhizoids a gravistimulation of the protonemata induces a transport of statoliths towards the tip. This transport is mainly along the cell axis and not parallel to the gravity vector. It is stressed that the sedimentation of statoliths in Chara rhizoids and protonemata as well as in gravity sensing cells in mosses and higher plants is accompanied by statolith movements based on interactions with the cytoskeleton. In tip-growing cells these movements direct the statoliths to a definite region of the cell where they can sediment and elicit a gravitropic curvature. In the statocytes of higher plants the interactions of the statoliths with the cytoskeleton probably do not serve primarily to move the statoliths but to transduce mechanical stresses from the sedimenting statoliths to the plasma membrane.     

Influence of gravity on the photomorphism of secondary moss protonemata

In dark-grown plantlets of the moss, Pottia intermedia, negatively gravitropic secondary protonemata differentiate from the superficial cells of leafy shoots. When transferred to the light, distal parts of the protonemata nearest to the apical cells begin to ramify and the apical cells of the side branches as well as of the main protonemal filaments often differentiate as buds. Dark-grown protonemata were oriented horizontally and illuminated from below with white light of different intensities. Only light with an intensity of 4.5 μmol·m⁻²·s⁻¹ was sufficient to induce: (a) phototropism in the apical cells, (b) light-directed initiation of branch primordia, and (c) directed growth of side branches and bud differentiation. Apical cells illuminated with light of lower (0.03–0.37 μmol·m⁻²·s⁻¹) intensity grew upwards (i.e., away from the light). It was shown that this upward growth was determined by the action of gravity. Although initiation of branch primordia was only slightly affected, their growth was strongly stimulated on the upper side of the protonemata.     

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.     

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.     

Operations of a spaceflight experiment to investigate plant tropisms

Plants will be an important component in bioregenerative systems for long-term missions to the Moon and Mars. Since gravity is reduced both on the Moon and Mars, studies that identify the basic mechanisms of plant growth and development in altered gravity are required to ensure successful plant production on these space colonization missions. To address these issues, we have developed a project on the International Space Station (ISS) to study the interaction between gravitropism and phototropism in Arabidopsis thaliana. These experiments were termed TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS) in 2006. In this paper, we provide an operational summary of TROPI and preliminary results on studies of tropistic curvature of seedlings grown in space. Seed germination in TROPI was lower compared to previous space experiments, and this was likely due to extended storage in hardware for up to 8 months. Video downlinks provided an important quality check on the automated experimental time line that also was monitored with telemetry. Good quality images of seedlings were obtained, but the use of analog video tapes resulted in delays in image processing and analysis procedures. Seedlings that germinated exhibited robust phototropic curvature. Frozen plant samples were returned on three space shuttle missions, and improvements in cold stowage and handing procedures in the second and third missions resulted in quality RNA extracted from the seedlings that was used in subsequent microarray analyses. While the TROPI experiment had technical and logistical difficulties, most of the procedures worked well due to refinement during the project.     

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.     

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.     

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.     

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."