An active role of the amyloplasts and nuclei of root statocytes in graviperception.

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception.     

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.     

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.     

Automorphosis of higher plants on a 3-D clinostat.

On a three-dimensional (3-D) clinostat, various plant organs developed statocytes capable of responding to the gravity vector. The graviresponse of primary roots of garden cress and maize grown on the clinostat was the same as the control roots, whereas that of maize coleoptiles was reduced. When maize seedlings were grown in the presence of 10(-4) M gibberellic acid and kinetin, the graviresponse of both roots and shoots was suppressed. The corresponding suppression of amyloplast development was observed in the clinostatted and the hormone-treated seedlings. Maize roots and shoots showed spontaneous curvatures in different portions on the 3-D clinostat. The hormone treatment did not significantly influence such an automorphic curvature. When the root cap was removed, maize roots did not curve gravitropically. However, the removal suppressed the automorphic curvatures only slightly. On the other hand, the removal of coleoptile tip did not influence its graviresponse, whereas the spontaneous curvature of decapitated coleoptiles on the clinostat was strongly suppressed. Also, cytochalasin B differently affected the gravitropic and the automorphic curvatures of maize roots and shoots. From these results it is concluded that the graviperception and the early processes of signal transmission are unnecessary for automorphoses under simulated microgravity conditions. Moreover, the results support the view that the amyloplasts act as statoliths probably via an interaction with microfilaments.     

Gravisensing in roots.

The mode of gravisensing in higher plants is not yet elucidated. Although, it is generally accepted that the amyloplasts (statoliths) in the root cap cells (statocytes) are responsible for susception of gravity. However, the hypothesis that the whole protoplast acts as gravisusceptor cannot be dismissed. The nature of the sensor that is able to transduce and amplify the mechanical energy into a biochemical factor is even more controversial. Several cell structures could potentially serve as gravireceptors: the endoplasmic reticulum, the actin network, the plasma membrane, or the cytoskeleton associated with this membrane. The nature of the gravisusceptors and gravisensors is discussed by taking into account the characteristics of the gravitropic reaction with respect to the presentation time, the threshold acceleration, the reciprocity rule, the deviation from the sine rule, the movement of the amyloplasts, the pre-inversion effect, the response of starch free and intermediate mutants and the effects of cytochalasin treatment. From this analysis, it can be concluded that both the amyloplasts and the protoplast could be the gravisusceptors, the former being more efficient than the latter since they can focus pressure on limited areas. The receptor should be located in the plasma membrane and could be a stretch-activated ion channel.     

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.     

Autonomic straightening of gravitropically curved cress roots in microgravity.

The typical response of plant organs to gravistimulation is differential growth that leads to organ bending. If the gravitropic stimulus is withdrawn, endogenous compensation of the graviresponse and subsequent straightening occur in some plants. For instance, autonomic straightening of Lepidium roots occurs when gravitropically-curved rootsare rotated on a clinostat (Stankovi et al., 1998a). To determine whether endogenous compensation of the graviresponse also occurs in space, microgravity-grown cress roots were laterally centrifuged in-flight and then returned to microgravity using Biorack hardware on a shuttle mission (STS-81). The cress roots were centrifuged at 4 different g-doses (0.1 x g and 1 x g for 15 or 75 min). All four treatments yielded varying degrees of root curvature. Upon removal from the centrifuge, roots in all four treatments underwent subsequent straightening in microgravity. This straightening resulted from a loss of gravitropic curvature in older regions of the root and the coordinated alignment of new growth. These results show that both microgravity and clinostat rotation on Earth are equivalent in stimulus withdrawal with respect to the induction of endogenous compensation of the curvature. Cress roots are the only plant organ shown to undergo compensation of the curvature in both microgravity and on a clinostat. The compensation of graviresponse in space rules out the hypothesis that the endogenous root straightening ("autotropism") represents a commitment to a pre-stimulus orientation with respect to gravity and instead suggests that there is a default tendency towards axiality following a withdrawal of a g-stimulus.     

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

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

Transduction of the gravity stimulus in the root statocyte.

The amyloplasts of root statocytes are considered to be the perceptors of gravity. However, their displacement and the starch they contain are not required for gravisensing. The mechanism of the transduction of gravistimulus remains therefore controversial. It is well known that the amplitude of the stimulus is dependent upon the intensity of the acceleration and the inclination of the root with respect to gravity. This strongly supports the hypothesis that the stimulus results in a mechanical effect (pressure or tension) on a cellular structure. Three cellular components are proposed as possible candidates for the role of transducer: the actin filaments, the endoplasmic reticulum and the plasma membrane with its ion channels. Recent results obtained in the frame of the IML 1 Mission of Spacelab show that the endoplasmic reticulum should rather be responsible for the termination of the stimulus. The contacts of amyloplasts with the distal ER could therefore be involved in the regulation of root growth.     

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.     

The effects of HGMFs on the plant gravisensing system.

High Gradient Magnetic Fields (HGMFs) offer new opportunities for studying the gravitropic system of plants. However, it is necessary to analyze the influence that HGMF can have on cellular processes and structures that may not be related to amyloplasts displacement. This paper considers possible HGMF effects on plants, which may accompany HGMF stimulation of amyloplasts and contribute to the mechanisms of the HGMF-induced curvature.     

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

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

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

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

Role of calcium in gravity perception of plant roots

Calcium ions may play a key role in linking graviperception by the root cap to the asymmetric growth which occurs in the elongation zone of gravistimulated roots. Application of calcium-chelating agents to the root cap inhibits gravitropic curvature without affecting growth. Asymmetric application of calcium to one side of the root cap induces curvature toward the calcium source, and gravistimulation induces polar movement of applied ⁴⁵Ca²⁺ across the root cap toward the lower side. The action of calcium may be linked to auxin movement in roots since 1) auxin transport inhibitors interfere both with gravitropic curvature and gravi-induced polar calcium movement and 2) asymmetric application of calcium enhances auxin movement across the elongation zone of gravistimulated roots. Indirect evidence indicates that the calcium-modulated regulator protein, calmodulin, may be involved in either the transport or action of calcium in the gravitropic response mechanism of roots.     

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.     

Growth of pea epicotyl in low magnetic field implication for space research

A magnetic field is an inescapable environmental factor for plants on the earth. However, its impact on plant growth is not well understood. In order to survey how magnetic fields affect plant, Alaska pea seedlings were incubated under low magnetic field (LMF) and also in the normal geo-magnetic environment. Two-day-old etiolated seedlings were incubated in a magnetic shield box and in a control box. Sedimentation of amyloplasts was examined in the epicotyls of seedlings grown under these two conditions. The elongation of epicotyls was promoted by LMF. Elongation was most prominent in the middle part of the epicotyls. Cell elongation and increased osmotic pressure of cell sap were found in the epidermal cells exposed to LMF. When the gravitational environment was 1G, the epicotyls incubated under both LMF and normal geomagnetic field grew straight upward and amyloplasts sedimented similarly. However, under simulated microgravity (clinostat), epicotyl and cell elongation was promoted. Furthermore, the epicotyls bent and amyloplasts were dispersed in the cells in simulated microgravity. The dispersion of amyloplasts may relate to the posture control in epicotyl growth under simulated microgravity generated by 3D clinorotation, since it was not observed under LMF in 1G. Since enhanced elongation of cells was commonly seen both at LMF and in simulated microgravity, all elongation on the 3D-clinostat could result from pseudo-low magnetic field, as a by-product of clinorotation. (i.e., clinostat results could be based on randomization of magnetic field together with randomization of gravity vector.) Our results point to the possible use of space for studies in magnetic biology. With space experiments, the effects of dominant environmental factors, such as gravity on plants, could be neutralized or controlled for to reveal magnetic effects more clearly.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

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

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

Gravisensitivity of cress roots.

The minimum dose (stimulus x time [gs]) eliciting a visible gravitropic response, has been determined using continuous and intermittent stimulation and two different accelerations at 1 g and 0.l g. The minimum dose of 20-30 gs estimated for microgravity roots and of 50-60 gs for roots grown on a 1 g-centrifuge indicated a higher sensitivity of microgravity roots. Applying intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus free interval of 118 s. The curvature of microgravity-grown roots to lateral stimulation by 0.1 g was remarkably smaller than by 1g in spite of the same doses which were applied to the seedlings. Microscopic investigations corresponding to stimulations in the range of the threshold values, demonstrated small displacement (< 2 micrometers) of statoliths in root statocytes. Accepting the statolith theory, one can conclude that stimulus transformation has to occur in the cytoplasm in close vicinity to the statoliths and that this transformation system was affected during seedling cultivation in microgravity.     

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.