Analysis of growth patterns during gravitropic curvature in roots ofZea mays by use of a computer-based video digitizer

A computer-based video digitizer system is described which allows automated tracking of markers placed on a plant surface. The system uses customized software to calculate relative growth rates at selected positions along the plant surface and to determine rates of gravitropic curvature based on the changing pattern of distribution of the surface markers. The system was used to study the time course of gravitropic curvature and changes in relative growth rate along the upper and lower surface of horizontally-oriented roots of maize (Zea mays L.). The growing region of the root was found to extend from about 1 mm behind the tip to approximately 6 mm behind the tip. In vertically-oriented roots the relative growth rate was maximal at about 2.5 mm behind the tip and declined smoothly on either side of the maximum. Curvature was initiated approximately 30 min after horizontal orientation with maximal (50°) curvature being attained in 3 h. Analysis of surface extension patterns during the response indicated that curvature results from a reduction in growth rate along both the upper and lower surfaces with stronger reduction along the lower surface.     

Gravitropic curvature of maize roots is not preceded by rootcap asymmetry

We tested whether the first response to gravistimulation is an asymmetry in the root tip that results from differential growth of the rootcap itself. The displacement of markers on the rootcap surface of maize (Zea mays L. cv. Merit) roots was quantified from videotaped images using customized software. The method was sensitive enough to detect marker displacements down to 15 microns and root curvature as early as 8 min after gravistimulation. No differential growth of the upper and lower sides of the cap occurred before or during root curvature. Fewer than a third of all gravistimulated roots developed an asymmetrical outline of the root tip after curvature had started, and this asymmetry did not occur in the rootcap itself. Our data support the view that the regions of gravitropic sensing and curvature are spatially separate during all phases of gravitropism in maize roots.     

Gravitropism: Interaction of Sensitivity Modulation and Effector Redistribution

Our increasing capabilities for quantitative hormone analysis and automated high resolution growth studies have allowed a reassessment of the classical Cholodny-Went hypothesis of gravitropism. According to this hypothesis, gravity induces redistribution of auxin toward the lower side of the organ and this causes the growth asymmetry that leads to reorientation. Arguments against the Cholodny-Went hypothesis that were based primarily on concerns over the timing and magnitude of the development of hormone asymmetry are countered by recent evidence that such asymmetry develops early and is sufficiently large to account for curvature. Thus, it appears that the Cholodny-Went hypothesis is fundamentally valid. However, recent comparative studies of the kinetics of curvature and the timing of the development of hormone asymmetry indicate that this hypothesis alone cannot account for the intricacies of the gravitropic response. It appears that time-dependent gravity-induced changes in hormone sensitivity as well as changes in sensitivity of the gravity receptor play important roles in the response.     

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.     

Analysis of growth during geotropic curvature in seedling hypocotyls

Abstract. The patterns of growth in organs curving under the influence of gravity were analysed by time-lapse photography of cress and cucumber hypocotyls which were delimited into 1 mm zones by ion-exchange beads. Geotropic curvature resulted from changes in growth rate on both sides of the organ. Growth inhibition of varying degrees of intensity occurred in all the previously growing zones of the upper (concave) side. An absolute reduction in length due to compression frequently occurred in some zones. Also, in both species growth stimulation was observed on the lower (convex) side. The disparity in growth rate between the upper and lower surfaces varied with time, being more apparent in the subapical region in the first hour of curvature. A later promotion of growth rate on the lower surface subsequently increased the curvature of the more basal zones. Autotropic straightening occurred as a consequence of growth changes, both inhibitory and stimulatory, in the apical zones. These events indicate a polarity of response in which apical zones have precedence over basal zones.     

A major factor in gravitropism in radish hypocotyls is the suppression of growth on the upper side of hypocotyls

The changes in length on the two opposite sides of etiolated radish (Raphanus sativus) hypocotyls prior to, and following gravitropic stimulation, were measured using an infrared-imaging system. It was observed that the growth suppression on the upper side began first at least 10 min after the onset of gravitropic stimulation, and after 30 min the acceleration in growth on the lower side started. The gravitropic curvature was steadily induced from 10 min. When radish hypocotyls were switched from a vertical to horizontal position for different durations and then replaced to the vertical position, the growth suppression on the gravity-stimulated (upper) side was observed in all cases, but the acceleration in growth on the opposite (lower) side appeared only in continuously gravity-stimulated seedlings, although it occurred later than the growth suppression on the upper side. These results suggest that the suppression in growth on the upper side of the hypocotyls is a direct effect of gravitropic stimulation, but not the acceleration on the lower side. When 4-methylthio-3-butenyl isothiocyanate (4-MTBI), which has an inhibitory activity against radish hypocotyl growth, was applied on the one side of radish hypocotyls and then the 4-MTBI-applied side or opposite side was placed in a horizontal position, the former showed greater bending than the control, suggesting that the growth suppression on the upper side is enhanced and maintained with MTBI application there. In the latter case, the seedlings showed less bending than the control, suggesting a decrease in growth on the lower side with MTBI application. All the results suggest that gravitropism of radish hypocotyls may be caused by an increase in growth-inhibiting substance(s) induced with gravitropic stimulation in the upper side, inducing growth inhibition there.     

Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism

The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H⁺ secretion, α-(p-chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA-induced inhibition. These results indicate a close relationship between the acid-growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism.     

Auxin Asymmetry during Gravitropism by Tomato Hypocotyls

Gravitropic asymmetry of auxin was observed in hypocotyls of tomato (Lycopersicon esculentum Mill.) soon after horizontal placement: the ratio of apically supplied [³H]IAA collected from the lower sides to that from the upper sides was about 1.4 between 5 and 10 minutes. This was adequately early to account for the beginning of curvature. The auxin asymmetry ratio rose to about 2.5 between 20 and 25 minutes, and to 3.5 during the main phase of curvature. This compares reasonably well with the roughly 3.9 ratio for elongation on the lower side to elongation on the upper side that is the basis for the curvature. These data extend evidence that the Went-Cholodny theory for the mediation of tropisms is valid for dicot stems. Also consistent with the theory, an auxin asymmetry ratio of 2.5 was observed when wrong-way gravitropic curvature developed following application of a high level of auxin. In addition to reversing the asymmetry of elongation, the large supplement of auxin resulted in lower net elongation. Previous data established that ethylene is not involved in this decrease of growth as a function of increasing level of auxin.     

Two distinct regions of response drive differential growth in Vigna root electrotropism

Although exogenous electric fields have been reported to influence the orientation of plant root growth, reports of the ultimate direction of differential growth have been contradictory. Using a high‐resolution image analysis approach, the kinetics of electrotropic curvature in Vigna mungo L. roots were investigated. It was found that curvature occurred in the same root toward both the anode and cathode. However, these two responses occurred in two different regions of the root, the central elongation zone (CEZ) and distal elongation zone (DEZ), respectively. These oppositely directed responses could be reproduced individually by a localized electric field application to the region of response. This indicates that both are true responses to the electric field, rather than one being a secondary response to an induced gravitropic stimulation. The individual responses differed in the type of differential growth giving rise to curvature. In the CEZ, curvature was driven by inhibition of elongation, whereas curvature in the DEZ was primarily due to stimulation of elongation. This stimulation of elongation is consistent with the growth response of the DEZ to other environmental stimuli.     

Growth patterns and gravitropic curvature of radish hypocotyls

The rapid growth rate of radish cells makes the hypocotyl particularly valuable in research on growth phenomena and gravitropism. An examination of mean data regarding the growth of aetiolated radish seedlings not subjected to gravitropic stimulation showed that there was an increase in the growth rate from day 3 to day 5. When the hypocotyl was placed horizontally all zones showed an increase in growth of the lower surface of the hypocotyl and generally a decrease in the growth rate in the upper surface. The upper apical part of the gravistimulated hypocotyl grew almost the same as controls. In some cases, in both 4- and 5-day-old seedlings, in the upper median part there was a lower growth rate than in controls. The zones of growth during the development of the hypocotyl were different. Analysis of curvature showed that the growth rate of the different zones was a function of their location in the hypocotyl and that the rate of curvature was different in various parts of the hypocotyl.     

Effects of Ethylene on the Kinetics of Curvature and Auxin Redistribution in Gravistimulated Roots of Zea mays

We tested the involvement of ethylene in maize (Zea mays L.) root gravitropism by measuring the kinetics of curvature and lateral auxin movement in roots treated with ethylene, inhibitors of ethylene synthesis, or inhibitors of ethylene action. In the presence of ethylene the latent period of gravitropic curvature appeared to be increased somewhat. However, ethylene-treated roots continued to curve after control roots had reached their final angle of curvature. Consequently, maximum curvature in the presence of ethylene was much greater in ethylene-treated roots than in controls. Inhibitors of ethylene biosynthesis or action had effects on the kinetics of curvature opposite to that of ethylene, i.e. the latent period appeared to be shortened somewhat while total curvature was reduced relative to that of controls. Label from applied ³H-indole-3-acetic acid was preferentially transported toward the lower side of stimulated roots. In parallel with effects on curvature, ethylene treatment delayed the development of gravity-induced asymmetric auxin movement across the root but extended its duration once initiated. The auxin transport inhibitor, 1-N-naphthylphthalamic acid reduced both gravitropic curvature and the effect of ethylene on curvature. Since neither ethylene nor inhibitors of ethylene biosynthesis or action prevented curvature, we conclude that ethylene does not mediate the primary differential growth response causing curvature. Because ethylene affects curvature and auxin transport in parallel, we suggest that ethylene modifies curvature by affecting gravity-induced lateral transport of auxin, perhaps by interfering with adaptation of the auxin transport system to the gravistimulus.     

Differential changes in size distribution of xyloglucan in the cell walls of gravitropically responding Pisum sativum epicotyls

Growth-related change in the size distribution of hemicellulosic wall polymers during the gravitropic curvature response of intact pea (Pisum sativum L. cv Alaska) epicotyls was examined by gel-filtration chromatography. The gravitropic response was characterized by the appearance of curvature 20 to 30 min after horizontal placement, with 35 degrees of curvature attained by 80 min. Correlated with the onset of curvature, on the upper side of the epicotyl, there was a conspicuous transient increase in the abundance of relatively large hemicellulosic xyloglucan polymers, similar to increases previously found under conditions where diminished wall extensibility was expected. On the lower side there was a moderate, slower, and longer-term increase in abundance of small xyloglucan, similar to changes previously found in connection with auxin-stimulated growth responses. Both shifts occurred primarily in the epidermis. They appear to represent two coordinated physiological mechanisms contributing to differential growth.     

Modelling of root growth and bending in two dimensions

A special co-ordinate system is developed for modelling the gravitropic bending of plant roots. It is based on the Local Theory of Curves in differential geometry and describes, in one dimension, growth events that may actually occur in two, or even three, dimensions. With knowledge of the spatial distributions of relative elemental growth rates (RELELs) for the upper and lower flanks of a gravistimulated root, and also their temporal dependencies, it is possible to compute the development of curvature along the root and hence describe the time-course of gravitropic bending. In addition, the RELEL distributions give information about the velocity field and the basipetal displacement of points along the root's surface. According to the Fundamental Theorem of Local Curve Theory, the x and y co-ordinates of the root in its bending plane are then determined from the associated values of local curvature and local velocity. With the aid of this model, possible mathematical growth functions that correspond to biological mechanisms involved in differential growth can be tested. Hence, the model can help not only to distinguish the role of various physiological or biophysical parameters in the bending process, but also to validate hypotheses that make assumptions concerning their relative importance. However, since the model is constructed at the level of the organ and treats the root as a fluid continuum, none of the parameters relate to cellular behaviour; the parameters must instead necessarily apply to properties that impinge on the behaviour of the external boundary of the root.     

Characterization of the asymmetric growth of gravistimulated snapdragon spikes by stem and cell dimension analyses

Growth patterns of detached spikes of gravistimulated snapdragon (Antirrhinum majus L.) were analyzed in detail. The length increment of 5-mm marked subsections in the upper and lower flanks of the stem-bending zone was measured during gravistimulation using time-lapse photographs. At the onset of bending, a negative relative growth rate of the upper flank was detected, followed by increased relative growth rate in both lower and upper flanks. Consequently, a differential stem growth pattern was obtained during gravistimulation, which was significantly and specifically abolished by calcium antagonists reported previously to inhibit stem curvature of snapdragon. The differential growth patterns resulted from dynamic modifications of the cell dimensions in the epidermal and cortical stem layers. Bending started with both shrinking and widening of the epidermal cells and a parallel decrease in length and height of cortical cells at the upper stem flank. These changes were accompanied with a concomitant increase in length and height of the cortical cells on the lower stem flank, followed by a growth increase of epidermal cells. Our results suggest that both the epidermal and cortical cells play an important role in gravitropic shoot bending of snapdragon.     

Root hydrotropism of an agravitropic pea mutant, ageotropum

We have partially characterized root hydrotropism of an agravitropic pea mutant, ageotropum (from Pisum sativum L. cv. Weibull's Weitor), without interference of gravitropism. Lowering the atmospheric air humidity inhibited root elongation and caused root curvature toward the moisture-saturated substrate in ageotropum pea. Removal of root tips approximately 1.5 mm in length blocked the hydrotropic response. A computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to a greater inhibition of elongation on the humid side than the dry side of the roots. Similarly, gravitropic curvature of Alaska pea roots resulted from inhibition of elongation on the lower side of the horizontally placed roots, while the upper side of the roots maintained a normal growth rate. Gravitropic bending of Alaska pea roots was apparent 30 min after stimulation, whereas differential growth as well as curvature in positive root hydrotropism of ageotropum pea became visible 4–5 h after the continuous hydrostimulation. Application of 2,3,5-triiodobenzoic acid or ethyleneglycol-bis-( β -aminoethylether)-N,N,N',N'-tetraacetic acid was inhibitory to both root hydrotropism of ageotropum pea and root gravitropism of Alaska pea. Some mutual response mechanism for both hydrotropism and gravitropism may exist in roots, although the stimulusperception mechanisms differ from one another.     

Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls

The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using (3)H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.     

Spatio-temporal quantification of differential growth processes in root growth zones based on a novel combination of image sequence processing and refined concepts describing curvature production

Differential growth processes in root and shoot growth zones are governed by the transport kinetics of auxin and other plant hormones. While gene expression and protein localization of hormone transport facilitators are currently being unraveled using state-of-the-art techniques of live cell imaging, the quantitative analysis of growth reactions is lagging behind because of a lack of suitable methods. A noninvasive technique, based on digital image sequence processing, for visualizing and quantifying highly resolved spatio-temporal root growth processes was applied in the model plant Arabidopsis thaliana and was adapted to provide precise information on differential curvature production activity within the root growth zone. Comparison of root gravitropic curvature kinetics in wild-type and mutant plants altered in a facilitator for auxin translocation allowed the determination of differences in the location and in the temporal response of curvature along the growth zone between the investigated plant lines. The findings of the quantitative growth analysis performed here confirm the proposed action of the investigated transport facilitator. The procedure developed here for the investigation of differential growth processes is a valuable tool for characterizing the phenomenology of a wide range of shoot and root growth movements and hence facilitates elucidation of their molecular characterization.     

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.     

Kinetic studies on the redistribution of endogenous growth regulators in gravireacting plant organs

The kinetics of redistribution of endogenous indole-3-acetic acid, cis-abscisic acid and gibberellic acid (+gibberellin A₇) in gravistimulated plant organs were followed by immunoassay, during the latent period and the phase of gravitropic curvature. Whereas in maize coleoptile tips, endogenous indole-3-acetic acid accumulated in the lower half of the organ (ratio 65:35, in favour of the lower half) before bending occurred, it was not possible to detect any significant lateral asymmetry of any of the growth regulators assayed in gravitropically reacting root tips of Zea mays L. and Vicia faba L. nor in hypocotyls of Helianthus annus L. Also, no indication was obtained for an exchange of growth regulators between peripheral and central cell layers of the sunflower hypocotyl. Evidence is presented that changes in the properties of the epidermal or subepidermal cell layers located in the lower half of the horizontally placed sunflower hypocotyl are largely responsible for the gravitropic reaction. An alteration in the subcellular compartmentation of IAA may be involved in this process.     

The role of the epidermis and cortex in gravitropic curvature of maize roots

In order to determine the role of the epidermis and cortex in gravitropic curvature of seedling roots of maize (Zea mays L. cv. Merit), the cortex on the two opposite flanks was removed from the meristem through the growing zone; gravitropic curvature was measured with the roots oriented horizontally with the cut flanks either on the upper and lower side, or on the lateral sides as a wound control. Curvature was slower in both these treatments (53° in 5 h) than in intact roots (82°), but there was no difference between the two orientations in extent and rate of curvature, nor in the latent time, showing that epidermis and cortex were not the site of action of the growth-regulating signal. The amount of cortex removed made no difference in the extent of curvature. Curvature was eliminated when the endodermis was damaged, raising the possibility that the endodermis or the stele-cortex interface controls gravitropic curvature in roots. The elongation rate of roots from which just the epidermis had been peeled was reduced by 0.01 mM auxin (indole-3-acetic acid) from 0.42 to 0.27 mm h^-1, contradicting the hypothesis that only the epidermis responds to changes in auxin activity during gravistimulation. These observations indicate that gravitropic curvature in maize roots is not driven by differential cortical cell enlargement, and that movement of growth regulator(s) from the tip to the elongating zone is unlikely to occur in the cortex.Abbreviations df degrees of freedom - IAA indole-3-acetic acid