Mutations in the gravity persistence signal loci in Arabidopsis disrupt the perception and/or signal transduction of gravitropic stimuli

Gravity plays a fundamental role in plant growth and development, yet little is understood about the early events of gravitropism. To identify genes affected in the signal perception and/or transduction phase of the gravity response, a mutant screen was devised using cold treatment to delay the gravity response of inflorescence stems of Arabidopsis. Inflorescence stems of Arabidopsis show no response to gravistimulation at 4 degrees C for up to 3 h. However, when gravistimulated at 4 degrees C and then returned to vertical at room temperature (RT), stems bend in response to the previous, horizontal gravistimulation (H. Fukaki, H. Fujisawa, M. Tasaka [1996] Plant Physiology 110: 933-943). This indicates that gravity perception, but not the gravitropic response, occurs at 4 degrees C. Recessive mutations were identified at three loci using this cold effect on gravitropism to screen for gravity persistence signal (gps) mutants. All three mutants had an altered response after gravistimulation at 4 degrees C, yet had phenotypically normal responses to stimulations at RT. gps1-1 did not bend in response to the 4 degrees C gravity stimulus upon return to RT. gps2-1 responded to the 4 degrees C stimulus but bent in the opposite direction. gps3-1 over-responded after return to RT, continuing to bend to an angle greater than wild-type plants. At 4 degrees C, starch-containing statoliths sedimented normally in both wild-type and the gps mutants, but auxin transport was abolished at 4 degrees C. These results are consistent with GPS loci affecting an aspect of the gravity signal perception/transduction pathway that occurs after statolith sedimentation, but before auxin transport.     

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.     

Curvature in Arabidopsis inflorescence stems is limited to the region of amyloplast displacement

Gravitropic sensing in stems and stem-like organs is hypothesized to occur in the endodermis. However, since the endodermis runs the entire length of the stem, the precise site of gravisensing has been difficult to define. In this investigation of gravisensitivity in inflorescence stems of Arabidopsis, we positioned stems in a high gradient magnetic field (HGMF) on a rotating clinostat. Approximately 40% of the young, wild-type (WT) inflorescences, for all positions tested, curved toward the HGMF in the vicinity of the stem exposed to the field. In contrast, when the wedge was placed in the basal region of older inflorescence stems, no curvature was observed. As a control, the HGMF was applied to a starchless mutant, and 5% of the stems curved toward the field. Microscopy of the endodermis in the WT showed amyloplast displacement in the vicinity of the HGMF. Additional structural studies demonstrated that the basal region of WT stems experienced amyloplast displacement and, therefore, suggest this region is capable of gravity perception. However, increased lignification likely prevented curvature in the basal region. The lack of apical curvature after basal amyloplast displacement indicates that gravity perception in the base is not transmitted to the apex. Thus, these results provide evidence that the signal (and thus, response) resulting from perception in Arabidopsis inflorescence stems is spatially restricted.     

SGR1, SGR2, SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana.

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1-1, sgr2-1, and sgr3-1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1-1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2-1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3-1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.     

Computer-based video digitizer analysis of surface extension in maize roots

We used a video digitizer system to measure surface extension and curvature in gravistimulated primary roots of maize (Zea mays L.). Downward curvature began about 25 +/- 7 min after gravistimulation and resulted from a combination of enhanced growth along the upper surface and reduced growth along the lower surface relative to growth in vertically oriented controls. The roots curved at a rate of 1.4 +/- 0.5 degrees min-1 but the pattern of curvature varied somewhat. In about 35% of the samples the roots curved steadily downward and the rate of curvature slowed as the root neared 90 degrees. A final angle of about 90 degrees was reached 110 +/- 35 min after the start of gravistimulation. In about 65% of the samples there was a period of backward curvature (partial reversal of curvature) during the response. In some cases (about 15% of those showing a period of reverse bending) this period of backward curvature occurred before the root reached 90 degrees. Following transient backward curvature, downward curvature resumed and the root approached a final angle of about 90 degrees. In about 65% of the roots showing a period of reverse curvature, the roots curved steadily past the vertical, reaching maximum curvature about 205 +/- 65 min after gravistimulation. The direction of curvature then reversed back toward the vertical. After one or two oscillations about the vertical the roots obtained a vertical orientation and the distribution of growth within the root tip became the same as that prior to gravistimulation. The period of transient backward curvature coincided with and was evidently caused by enhancement of growth along the concave and inhibition of growth along the convex side of the curve, a pattern opposite to that prevailing in the earlier stages of downward curvature. There were periods during the gravitropic response when the normally unimodal growth-rate distribution within the elongation zone became bimodal with two peaks of rapid elongation separated by a region of reduced elongation rate. This occurred at different times on the convex and concave sides of the graviresponding root. During the period of steady downward curvature the elongation zone along the convex side extended farther toward the tip than in the vertical control. During the period of reduced rate of curvature, the zone of elongation extended farther toward the tip along the concave side of the root. The data show that the gravitropic response pattern varies with time and involves changes in localized elongation rates as well as changes in the length and position of the elongation zone. Models of root gravitropic curvature based on simple unimodal inhibition of growth along the lower side cannot account for these complex growth patterns.     

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.     

Spatial organization of the gravitropic response in plants: applicability of the revised local curvature distribution model to Triticum aestivum coleoptiles

The revised local curvature distribution model, which provides accurate computer simulations of the gravitropic response of mushroom stems, was found to produce accurate simulations of the gravitropic reaction of wheat ( Triticum aestivum ) coleoptiles. The key feature of the mathematical model that enables it to approach universality of application is the assumption that the stem has an autonomic straightening reaction (curvature compensation or 'autotropism'). In the model, the local bending rate for any segment of the organ is determined by the difference between the 'bending signal' (generated by the gravitropic signal perception system) and a 'straightening signal' (which is proportional to the local curvature of the segment). The model reveals three major differences between the gravitropic reactions of wheat coleoptiles and Coprinus mushroom stems. First, in Coprinus , the capacity for autonomic straightening is much more concentrated in the apical region of the stem. Second, local perception of the gravitropic signal, which is necessary for exact simulation in Coprinus , is not needed in wheat coleoptiles (the corresponding constant in the model can be set to zero). Third, the transmission rate of the gravitropic signal is about seven times faster in wheat coleoptiles than in the mushroom stem. Thus, we demonstrate that a single model, depending on the values given to its parameters, is able to simulate the spatial organization of the gravitropic reaction of wheat coleoptiles and Coprinus mushroom stems. The model promises to be a valuable predictive tool in guiding future research into the gravitropic reaction of axial organs of all types.     

A universal role for inositol 1,4,5-trisphosphate-mediated signaling in plant gravitropism

Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced beta-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants.     

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis . Previous studies have shown that the arc 6 and arc 12 ( a ccumulation and r eplication of c hloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc 6 and arc 12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc 6 and arc 12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc 6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.     

The role of amyloplasts during gravity perception in gynophores of the peanut plant (Arachis hypogaea).

Gravitropic perception and response are essential for the completion of the reproductive life cycle of the peanut plant (Arachis hypogaea L.). The developing seeds are buried in the soil by a specialized organ, the gynophore, allowing the fruit to mature underground. Controversy exists about the site of graviperception in the gynophore: previous workers suggested that the intercalary meristem was the zone where gravity was perceived. Taking the starch statolith hypothesis for graviperception as a framework, we explored the possibility that the starch-grain filled plastids (amyloplasts) in the starch sheath of the gynophore may be acting as gravisensors. We show that these amyloplasts sediment readily with respect to the gravity vector within 30 min of reorientation, and before there is a measurable gravitropic response. Gynophore explants were incubated with gibberellic acid and kinetin, in darkness, to remove starch from the amyloplasts. Destarching the gynophores did not inhibit overall growth of the organ, but reduced the gravitropic response curvature by 82% compared to water-treated controls. In addition, gynophores placed on a rotating clinostat (without hormone treatment) also showed a reduced gravitropic response. In conclusion, the evidence presented in this work strongly suggests that the amyloplasts of the starch sheath are responsible for gravitropic perception in the peanut gynophore. A model for graviperception in the gynophore is presented.     

Mathematical modelling of morphogenesis in fungi: a key role for curvature compensation ('autotropism') in the local curvature distribution model

The assumption that the mushroom stem has the ability to undergo autonomic straightening enables a mathematical model to be written that accurately mimics the gravitropic reaction of the stems of Coprinus cinereus . The straightening mechanism is called curvature compensation here, but is equivalent to the 'autotropism' that often accompanies the gravitropic reactions of axial organs in plants. In the consequently revised local curvature distribution model, local bending rate is determined by the difference between the 'bending signal' (generated by gravitropic signal perception systems) and the 'straightening signal' (proportional to the local curvature at the given point). The model describes gravitropic stem bending in the standard assay with great accuracy but has the virtue of operating well outside the experimental data set used in its derivation. It is shown, for example, that the mathematical model can be fitted to the gravitropic reactions of stems treated with metabolic inhibitors by a change of parameters that parallel the independently derived physiological interpretation of inhibitor action. The revised local curvature distribution model promises to be a predictive tool in the further analysis of gravitropism in mushrooms.     

Evidence for altered polar and lateral auxin transport in the gravity persistent signal (gps) mutants of Arabidopsis

Plant shoots do not respond when they are reoriented relative to gravity at 4 degrees C. However, when returned to vertical at room temperature, these organs bend in response to the previous cold gravistimulation. The inflorescence stem of the Arabidopsis thaliana gravity persistent signal (gps) mutants respond abnormally after the cold gravistimulation: gps1 does not bend when returned to room temperature, gps2 bends the wrong way and gps3 over-responds, curving past the predicted angle. In wild type and the mutants, basipetal auxin transport in the inflorescence stem was abolished at 4 degrees C but restored when plants were returned to room temperature. In gps1, auxin transport was increased; in both gps2 and gps3, no significant difference was found when compared to wild type. Expression of the auxin-inducible P(IAA2)::GUS reporter gene, indicated that auxin-induced gene expression was redistributed to the lower side of the inflorescence stem in wild type after gravistimulation at 4 degrees C. In gps1, no asymmetries in P(IAA2)::GUS expression were seen. In gps2, P(IAA2)::GUS expression was localized to the upper side of the stem and in gps3, asymmetric P(IAA2):GUS expression was extended throughout the elongation zone of the inflorescence stem. These results are consistent with altered lateral Indole-3-acetic-acid (IAA) gradients being responsible for the phenotype of each mutant.     

Spatial separation of light perception and growth response in maize root phototropism

Although the effects of gravity on root growth are well known and interactions between light and gravity have been reported, details of root phototropic responses are less documented. We used high-resolution image analysis to study phototropism in primary roots of Zea mays L. Similar to the location of perception in gravitropism, the perception of light was localized in the root cap. Phototropic curvature away from the light, on the other hand, developed in the central elongation zone, more basal than the site of initiation of gravitropic curvature. The phototropic curvature saturated at approximately 10 micromoles m-2 s-1 blue light with a peak curvature of 29 +/- 4 degrees, in part due to induction of positive gravitropism following displacement of the root tip from vertical during negative phototropism. However, at higher fluence rates, development of phototropic curvature is arrested even if gravitropism is avoided by maintaining the root cap vertically using a rotating feedback system. Thus continuous illumination can cause adaptation in the signalling pathway of the phototropic response in roots.     

Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.     

Early Gravi-Electrical Responses in Bean Epicotyls

The relationship between gravitropism and surface electrical potentials was studied using etiolated epicotyls of adzuki bean (Phaseolus angularis). Early downward curvature (or transient positive gravitropic response) was observed about 1 min after gravistimulation. The downward curvature was closely related to the speed of the subsequent upward curvature. Surface electrical potentials decreased cooperatively in a limited region on the upper side within only 0.5 to 2 min. This is the earliest event found so far to follow gravistimulation of intact epicotyls. The rapid change in the potential had a high correlation with the early downward curvature and also the subsequent negative gravitropism. It is suggested that the rapid potential change plays an important role in gravity perception.     

The endodermis and shoot gravitropism

Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgrl), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root(shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.     

Growth and gravitropism of corn roots in solution

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m⁻³ Ca²⁺. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.     

Actin cytoskeleton rearrangements during the gravitropic response of Arabidopsis roots

Gravitropic response is a plant growth response against changing its position relative to the gravity vector. In the present work we studied actin cytoskeleton rearrangements during Arabidopsis root gravitropic response. Two alternative approaches were used to visualize actin microfilaments: histochemical staining of fixed roots with rhodamine-phalloidin and live imaging of microfilaments in GFP-fABD2 transgenic plants. The curvature of actin microfilaments was shown to be increased within 30–60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. Methodological issues of actin cytoskeleton visualization in the study of Arabidopsis root gravitropic response, as well as the role of microfilaments at the stages of gravity perception, signal transduction and gravitropic bending formation are discussed. It is concluded that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed.