The effect of the external medium on the gravity-induced polarity of cytoplasmic streaming in Chara corallina (Characeae)

Gravity induces a polarity of cytoplasmic streaming in vertical internodal cells of Chara such that the downwardly directed stream moves faster than the upwardly directed stream. In order to determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best explain the gravity response in Chara internodal cells, we controlled the physical properties of the external medium, including density and osmolarity, with impermeant solutes and examined the effect on the polarity of cytoplasmic streaming. As the density of the external medium is increased, the polarity of cytoplasmic streaming decreases and finally disappears when the density of the external medium is equal to that of the cell (1015 kg/m3). A further increase in the density of the external medium causes a reversal of the gravity response. These results are consistent with the gravitational pressure theory of gravity sensing since the buoyancy of the protoplast is dependent on the difference between the density of the protoplast and the external medium, and are inconsistent with the statolith theory since the buoyancy of intracellular particles are unaffected by changes in the external medium.     

Cytochalasin D does not inhibit gravitropism in roots

It is generally thought that sedimenting plastids are responsible for gravity sensing in higher plants. We directly tested the model generated by the current statolith hypothesis that the gravity sensing that leads to gravitropism results from an interaction between the plastids and actin microfilaments. We find that the primary roots of rice, corn, and cress undergo normal gravitropism and growth even when exposed to cytochalasin D, a disruptor of actin microfilaments. These results indicate that an interaction between amyloplasts and the actin cytoskeleton is not critical for gravity sensing in higher plants and weaken the current statolith hypothesis.     

Graviresponsiveness of surgically altered primary roots of Zea mays

We examined the gravitropic responses of surgically altered primary roots of Zea mays to determine the route by which gravitropic inhibitors move from the root tip to the elongating zone. Horizontally oriented roots, from which a 1-mm-wide girdle of epidermis plus 2-10 layers of cortex were removed from the apex of the elongating zone, curve downward. However, curvature occurred only apical to the girdle. Filling the girdle with mucilage-like material transmits curvature beyond the girdle. Vertically oriented roots with a half-girdle' (i.e. the epidermis and 2-10 layers of the cortex removed from half of the circumference of the apex of the elongating zone) curve away from the girdle. Inserting the half-girdle at the base of the elongating zone induces curvature towards the girdle. Filling the half-circumference girdles with mucilage-like material reduced curvature significantly. Stripping the epidermis and outer 2-5 layers of cortex from the terminal 1.5 cm of one side of a primary root induces curvature towards the cut, irrespective of the root's orientation to gravity. This effect is not due to desiccation since treated roots submerged in water also curved towards their cut surface. Coating a root's cut surface with a mucilage-like substance minimizes curvature. These results suggest that the outer cell-layers of the root, especially the epidermis, play an important role in root gravicurvature, and the gravitropic signals emanating from the root tip can move apoplastically through mucilage.     

Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity

The minimum dose (dose = stimulus x time), one of three threshold values related to gravity, was determined under microgravity conditions for cress roots. Seedlings were cultivated on a 1g centrifuge in orbit and under microgravity, respectively. After continuous stimulation on a threshold centrifuge, minimum doses of 20-30 gs for microgravity roots and 50-60 gs for roots grown on a 1g centrifuge were estimated, which indicated that microgravity roots have a higher sensitivity than 1g roots. These results do not confirm the threshold value of 12gs which was determined for cress roots using the slow rotating clinostat. Following application of intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus-free interval of 118s. Generally, this demonstrates that higher plants are able to 'sum up' stimuli which are below the threshold value. Microscopic investigations of the cellular structure corresponding to stimulations in the range of the threshold value demonstrated a small displacement of statoliths in root statocytes. No significant correlation was observed between gravitropic curvature and statolith displacement. If the statolith theory is accepted, it can be concluded that stimulus transformation must occur in the cytoplasm in the near vicinity of the statoliths and that this transformation system--probably involving cytoskeletal elements--must have been affected during microgravity seedling cultivation.     

Signal transmission during gravitropic curvature of primary roots of Zea mays

Primary roots of Zea mays cv. Ageotropic are nonresponsive to gravity and elongate approximately 0.80 mm h^?1. Applying mucilage-like material (K-Y Jelly) to the terminal 1.5 cm of these roots induces graviresponsiveness and slow elongation 28% (i.e. from 0.80 to 0.58mm h^?1). Applying mucilage-like material to one side of the terminal 1.5 cm of the root induces curvature toward the mucilage, irrespective of the root's orientation to gravity. Applying a 2-mm-wideband of mucilage-like material to a root's circumference 8 to 10 mm behind the root cap neither induces gravicurvature nor affects elongation significantly. Similarly, applying mucilage-like material to only the root cap does not significantly affect elongation or graviresponsiveness. Gravicurvature of mutant roots occurs only when mucilage-like material is applied to the root/root-cap junction. Reversing the caps of wild-type and mutant roots produces gravitropic responses characteristic of the root cap rather than the host root. These results are consistent with the suggestion that gravitropic effectors are growth inhibitors that move apoplastically through mucilage between the root cap and root.     

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.     

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 μM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 μM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.     

Ethylene plays multiple nonprimary roles in modulating the gravitropic response in tomato.

Ethylene is known to interact with auxin in regulating stem growth, and yet evidence for the role of ethylene in tropic responses is contradictory. Our analysis of four mutants of tomato (Lycopersicon esculentum) altered in their response to gravity, auxin, and/or ethylene revealed concentration-dependent modulation of shoot gravitropism by ethylene. Ethylene inhibitors reduce wild-type gravicurvature, and extremely low (0.0005-0.001 microliter L-1) ethylene concentrations can restore the reduced gravitropic response of the auxin-resistant dgt (diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene inhibit the gravitropic response of all but the ethylene-insensitive nr (never-ripe) mutant. The gravitropic responses of nr and the constitutive-response mutant epi (epinastic) are slightly and significantly delayed, respectively, but otherwise normal. The reversal of shoot gravicurvature by red light in the lz-2 (lazy-2) mutant is not affected by ethylene. Taken together, these data indicate that, although ethylene does not play a primary role in the gravitropic response of tomato, low levels of ethylene are necessary for a full gravitropic response, and moderate levels of the hormone specifically inhibit gravicurvature in a manner different from ethylene inhibition of overall growth.     

Gravitropism in roots of intermediate-starch mutants of Arabidopsis

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated, by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as. determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the slarchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to that of the starchless mutant, it appears that 51–60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the 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.     

The Role of Starch in the Gravitropic Response of the Lentil Root

It is well accepted that the amyloplasts of the cap are responsible for gravisensing in primary roots. However, roots with starch-depleted plastids are able to respond to gravistimulus, but their curvature is slower than that of roots containing amyloplasts. The goal of our experiment was to analyse the effects of natural variations of statolith starch in the gravitropic response of lentil roots to a stimulation in the horizontal position. In lentil seedlings grown in the vertical position for 26 h, the volume of the amyloplasts in the statocytes differed between individual roots. The amount of starch in the cap was determined parallel to the rate of gravitropic curvature. There was no statistical correlation between the intensity of the gravitropic response and the starch content in the statocytes. Lentil roots were treated with gibberellic acid (GA₃) at 32°C in order to reduce the volume of starch in the statoliths. There was 53% less starch in the cap of GA₃treated roots as compared to the cap of control roots. But there was no relationship between starch content in the cap and the responsiveness of the root to a gravistimulus, except when the amount of starch was small.     

Effect of Inhibitors of Auxin Transport and of Calmodulin on a Gravisensing-Dependent Current in Maize Roots

Some characteristics of the gravity sensing mechanism in maize root caps were investigated using a bioelectric current as an indicator of gravity sensing. This technique involves the measurement of a change in the current density which arises at the columella region coincidently with the presentation time. Two inhibitors of auxin transport, triiodobenzoic acid and naphthylphthalamic acid, blocked gravitropic curvature but not the change in current density. Two inhibitors of calmodulin activity, compound 48/80 and calmidazolium, blocked both curvature and gravity-induced current. The results suggest that auxin transport is not a component of gravity sensing in the root cap. By contrast, the results suggest that calmodulin plays an intrinsic role in gravity sensing.     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Modification of the gravitropic response of seedling roots of raneseed (Brassica napus) transformed by Agrobacterium rhizogenes A4

We have examined the growth and gravitropic response of seedling roots of rapeseed ( Brassica napus . CrGC5–1) transformed by Agrobacterium rhizogenes A4, in order to evaluate if this could constitute a new model system for the study of gravitropism. The transformed clone chosen for study had integrated full-length TL- and TR-DNA from pRi (the root inducing plasmid), and thus included all of the agrobacterial genes potentially involved in the modified phenotype of transformed plants. In the vertical position, the growth rate of transformed roots was higher than controls. During 24 h of continuous stimulation, the optimal angle for gravitropic bending in normal roots was 135° (with respect to the gravity axis), with decreasing response at 90° and 45°. For transformed roots, slight curvature developed at 45° and at 90°, and stronger curvature was observed at 135°, though transformed roots tips never reached the vertical position. The minimum stimulation time necessary to elicit a response (presentation time) was also determined: it was signficantly shorter in normal roots (80 s) than in transformed ones (120 s). The results show that pRi transformed roots are less sensitive to gravity than normal roots.     

Sequence of key events in shoot gravitropism

It has recently been shown that asymmetric acid efflux is closely correlated with the gravitropic curvature of plant shoots and roots. The research reported here addresses whether auxin (IAA) redistribution in shoots is the cause or result of asymmetric acid efflux.

When abraded sunflower (Helianthus annuus cv Mammoth) hypocotyls are submerged in 20 millimolar neutral buffer, gravicurvature is greatly retarded relative to 0.2 millimolar controls. Nevertheless, in both buffer systems there is a similar redistribution of [³H]IAA toward the lower surface of gravistimulated sunflower hypocotyls. These results suggest that graviperception initiates IAA redistribution, which in turn results in auxin-induced asymmetric H⁺ efflux across the shoot. This interpretation is reinforced by data showing the effects of removal of the epidermal layers (peeling), osmotic shock, and morphactin treatment on gravicurvature and [³H]IAA redistribution. Peeling and osmotic shock inhibit gravicurvature but not redistribution. Morphactin inhibits both processes but does not inhibit hypocotyl straight growth.

     

Ethylene Interacts with Auxin in Regulating Developmental Attenuation of Gravitropism in Flax Root

Previous research shows that gravity-sensing in flax (Linum usitatissimum) root is initiated during seed imbibition and precedes root emergence. In this study we investigated the developmental attenuation of flax root gravitropism post-germination and the involvement of ethylene. Gravity response deteriorated significantly from 3 to 11?h after root emergence, which occurred at around 19?h after imbibition (that is, from “age” 22 to 30?h). Although the root elongation rate increased from 22 to 30?h, the gravitropic curving rate declined steadily. Older roots were able to tolerate higher levels of exogenous IAA before inhibition of elongation and gravitropism occurred. The age-dependent effect of IAA on root growth and gravitropism suggests that young roots are more sensitive to auxin and respond to a smaller vertical auxin gradient than older roots upon horizontal gravistimulation. The ethylene synthesis inhibitor AVG (2-aminoethoxyvinyl glycine, 10?μM) or ethylene action inhibitor Ag⁺ (10?μM) stimulated gravitropic curvature of 30?h roots by 24 and 32%, respectively, but had no effect on 22?h roots, suggesting that as roots age, ethylene begins to play a role in root gravitropism. The auxin transport inhibitor NPA (N-naphthylphthalamic acid, 50?μM) reduced gravitropic curvature of 30?h roots by 24% but had no effect on 22?h roots. On the other hand, treating roots simultaneously with the auxin transport inhibitor and ethylene synthesis or action inhibitor stimulated gravitropic curvature of 30?h roots but not 22?h roots. Taken together, these data indicate that as roots develop, their weakened gravity response is due to decreased auxin sensitivity and possibly auxin transport regulated by ethylene.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

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.     

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.     

The Arabidopsis WAVY GROWTH 2 protein modulates root bending in response to environmental stimuli

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an alpha/beta-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.