Thigmotropism and the modulation of tropistic curvature by mechanical perturbation in cucumber hypocotyls

Mechanical perturbation (MP) applied unilaterally to cucumber ( Cucumis sativus L.) hypocotyls induced thigmotropic curvature toward the stimulus. Gravitropic or phototropic curvature of the hypocotyl was inhibited by symmetrical application of MP to both sides of the hypocotyl. When both MP and IAA were unilaterally applied simultaneously to the same side, the hypocotyls always bent toward the MP stimulus, as in thigmotropism alone. Thus, the exogenous IAA did not control the direction of curvature. Aminoethoxyvinyl glycine (AVG) blocked thigmotropism as well as gravitropism and phototropism, but promoted IAA-induced curvature. MP-stimulated ethylene evolution peaked about 4 h after MP, followed by a peak of thigmotropic curvature. For all tropisms more ethylene evolved from the stimulated side than from the other side of the hypocotyls. MP-induced ethylene acting as a growth inhibitor, auxin-transport inhibitor, and/or modulator of tissue sensitivity to auxin, may be involved in thigmotropism and MP-induced inhibition of various tropisms. Ethylene produced as a result of MP was not affected by the removal of cotyledons. This MP-induced ethylene was additive to that of phototropically or gravitropically stimulated ethylene.     

Geotropic response of cucumber hypocotyls

Exogenous auxin applied to the apical bud of intact cucumberseedlings promoted elongation of the hypocotyl, mostly of theapical zone (zone I) and to a lesser extent, the middle andbasal zones (zones II and III). Geotropic curvature was clearlyobserved 30 min after geostimulation. The curvature began atzone I and moved toward the basal zones with prolonged geostimulation.Decapitation (removal of apex and cotyledons) did not produceany appreciable effect on the geotropic response, but the curvaturewas reduced with the removal of zone I. Auxin application toseedlings without zone I restored their ability to respond togravity. The lower epidermis of horizontally oriented hypocotylsplayed a more important role than the upper in the geotropicresponse. The extensibility of the lower epidermal cell wallwas increased significantly by the geostimulus before the onsetof the curvature response. (Received August 24, 1973; )     

Polymer mobility in cell walls of cucumber hypocotyls

Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.     

Magnetophoretic induction of curvature in coleoptiles and hypocotyls

Coleoptiles of barley (Hordeum vulgare) were positioned in a high gradient magnetic field (HGMF, dynamic factor gradient of H(2)/2 of 10(9)-10(10) Oe2 cm-1), generated by a ferromagnetic wedge in a uniform magnetic field and rotated on a 1 rpm clinostat. After 4 h 90% of coleoptiles had curved toward the HGMF. The cells affected by HGMF showed clear intracellular displacement of amyloplasts. Coleoptiles in a magnetic field next to a non-ferromagnetic wedge showed no preferential curvature. The small size of the area of nonuniformity of the HGMF allowed mapping of the sensitivity of the coleoptiles by varying the initial position of the wedge relative to the coleoptile apex. When the ferromagnetic wedge was placed 1 mm below the coleoptile tip only 58% of the coleoptiles curved toward the wedge indicating that the cells most sensitive to intracellular displacement of amyloplasts and thus gravity sensing are confined to the top 1 mm portion of barley coleoptiles. Similar experiments with tomato hypocotyls (Lycopersicum esculentum) also resulted in curvature toward the HGMF. The data strongly support the amyloplast-based gravity-sensing system in higher plants and the usefulness of HGMF to substitute gravity in shoots.     

Magnetophoretic induction of curvature in coleoptiles and hypocotyls

Coleoptiles of barley (Hordeum vulgare) were positioned in ahigh gradient magnetic field (HGMF, dynamic factor H²/2 of 10⁹–10¹⁰Oe² cm^–1), generated by a ferromagnetic wedge in a uniformmagnetic field) and rotated on a 1 rpm clinostat. After 4 h90% of coleoptiles had curved toward the HGMF. The cells affectedby HGMF showed clear intracellular displacement of amyloplasts.Coleoptiles in a magnetic field next to a non-ferromagneticwedge showed no preferential curvature. The small size of thearea of non-uniformity of the HGMF allowed mapping of the sensitivityof the coleoptiles by varying the initial position of the wedgerelative to the coleoptile apex. When the ferromagnetic wedgewas placed 1 mm below the coleoptile tip only 58% of the coleoptilescurved toward the wedge indicating that the cells most sensitiveto intracellular displacement of amyloplasts and thus gravitysensing are confined to the top 1 mm portion of barley coleoptiles.Similar experiments with tomato hypocotyls Lycopersicum esculentum)also resulted in curvature toward the HGMF. The data stronglysupport the amyloplast-based gravity-sensing system in higherplants and the usefulness of HGMF to substitute gravity in shoots. Key words: Avena sativa, Hordeum vulgare, Lycopersicon esculentum, curvature, gravitropism, high gradient magnetic field, magnetophoresis     

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.     

IAA-oxidase activity in light grown cucumber hypocotyls treated with helminthosporol

Summary Helminthosporol (H-ol) promoted the growth of light grown cucumber hypocotyls. The IAA-oxidase activity per dry weight of the hypocotyl of H-ol treated materials was less than that of controls. There was an inverse relationship between IAA-oxidase activity and H-ol induced elongation of the hypocotyl. However, when IAA-oxidase activity per plant was compared between H-ol treated materials and controls there was no essential difference. It is concluded that the apparent inhibitory effect of H-ol on IAA-oxidase activity is not directly related to the mechanism of H-ol action.     

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.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.     

Analysis of growth during phototropic curvature of cress hypocotyls

Abstract. Growth rates throughout an organ curving phototropically under continuous, unilateral while light were monitored by lime-lapse photography of cress hypocotyls marked into 1 mm sections by two rows of ion-exchange beads. Curvature resulted from an integrated sequence of changes in growth rate on each side of the organ, but the actual patterns of change and, therefore rales of curvature, differed within even this one species, depending upon the immediate pretreatment of the seedlings. Transference of seedlings from darkness to unilateral irradiation gave immediate growth inhibition on both sides of the organ. Curvature resulted from differential recovery of growth rate, being seen first on the shaded side, most prominently in the apical regions; only 2h after initial exposure to light did growth recover on the lit (lower) side. On the other hand, transfer of seedlings from omnilateral to unilateral irradiation of the same intensity resulted in simultaneous growth inhibition on the irradiated side and stimulated growth on the shaded side: this growth stimulation of the shaded side was greater than occurred in totally darkened control plants.     

Gravitropism of cucumber hypocotyls: biophysical mechanism of altered growth

Abstract. The biophysical basis for the changes in cell elongation rate during gravitropism was examined in aetiolated cucumber ( Cucumis sativus L.) hypocotyls. Bulk osmotic pressures on the two sides of the stem and in the epidermal cells were not altered during the early time course of gravitropism. By the pressureprobe technique, a small increase in turgor (0.3 bar, 30 kPa) was detected on the upper (inhibited) side, whereas there was a negligible decrease in turgor on the lower (stimulated) side. These small changes in turgor and water potential appeared to be indirect, passive consequences of the altered growth and the small resistance for water movement from the xylem, and indicated that the change in growth was principally due to changes in wall properties. The results indicate that the hydraulic conductance of the watertransport pathway was large (.25 h ¹ bar ¹) and the water potential difference supporting cell expansion was no greater than 0.3 bar (30 kPa). From pressureblock experiments, it appeared that upon gravitropic stimulation (1) the yield threshold of the lower half of the stem did not decrease and (2) the wall on the upper side of the stem was not made more rigid by a cross-linking process. Mechanical measurements of the stress/strain properties of the walls showed that the initial development of gravitropism did not involve an alteration of the mechanical behaviour of the isolated walls. Thus, gravitropism in cucumber hypocotyls occurs principally by an alteration of the wall relaxation process, without a necessary change in wall mechanical properties.     

Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection

The induction of peroxidases (EC 1.11.1.7) during elicitation of lignification by α-1,4-linked oligogalacturonides in cucumber hypocotyl segments ( Cucumis sativus L. cv, Wisconsin SMR 58) was investigated. The wounding associated with the preparation of hypocotyl segments induced a 19-fold increase in peroxidase activity during the following 72 h. The increase was partially due to an increase in activity of a constitutive peroxidase with a pI of 8.9 and partially due to the expression of new peroxidase isozymes with pIs of 3.8, 5.4, 6.2, 9.1 and 9.4. The oligogalacturonides did not induce any peroxidase activity in addition to the wound-induced activity. These results suggest that either the constitutive peroxidase isozyme (pI 8.9) of intact hypocotyls or some of the wound-induced peroxidases are involved in the oligogalacturonide-induced lignification.
Induction of the peroxidases by wounding was inhibited by cycloheximide. This indicates that they accumulate as a result of de novo protein synthesis. Actinomycin D caused only a modest inhibition of the wound-induction peroxidases, indicating that the process is regulated at the level of translation.
Peroxidase activity increased more rapidly in resistant than in susceptible cucumber hypocotyls after inoculation with the pathogen Cladosporium cucumerinum Ellis & Arthur. The pattern of isozymes which was induced by fungal infection of resistant hypocotyls was similar to the pattern of isozymes induced by wounding. This suggests that similar induction mechanisms may be involved in the two processes.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasahara et al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+2O g) but not under acropetal hypergravity (H-13 g), as compared to growth under the control conditions (C+1 g and C-1 g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Wall extensibility and gravitropic curvature of sunflower hypocotyls: correlation between timing of curvature and changes in extensibility

Abstract. Gravitropic curvature results from unequal growth rates on the upper and lower sides of horizontal stems. These unequal growth rates cold be due to differences in wall extensibility between the two sides. To test this, the time course of curvature of horizontal sunflower ( Helianthus anmus L.) hypocotyls was determined and compared with the time courses of changes in Instron-measured wall extensibility (PEx) of the upper and lower epidermal layers. As gravicurvature developed, so did the difference in PEx between the upper and lower epidermis. The enhanced growth rate on the lower side during the period of maximum increase in curvature was matched by PEx values greater than those of the vertical control, while the inhibited growth rate on the upper side was accompanied by PEx values below that of the control. The close correlation between changes in growth rates and alterations in PEx demonstrates that changes in wall extensibility play a major role in controlling gravicurvature.     

Quantification of curvature production in cylindrical organs, such as roots and hypocotyls

Differential growth curvature rate (DGCR), defined as the spatial derivative of the tropic speed, was derived as a measure of curvature production in cylindrical organs. Its relation to usual concepts, such as curvature (kappa), rate of curvature (dkappa/dt) and differential growth profiles, was determined. A root gravitropism model, testing the hypothesis of one and two motors, exemplified its capabilities.DGCR was derived using cylindrical geometry and its meaning was obtained through a curvature conservation equation. The root gravitropism model was solved using a discrete difference method on a computer.DGCR described curvature production independently of growth, and was superior to dkappa/dt, which underestimated production. Moreover, DGCR profiles were able to differ between one and two motors, while profiles of kappa and dkappa/dt were not.The choice of the measure of curvature production has a large impact on experimental results, in particular when spatial and temporal patterns of differential growth need to be determined. DGCR was shown to fulfill the accuracy needed in the quantification of curvature production and should thus serve as a helpful tool for measurements.     

Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue-light irradiation

Rapid suppression of hypocotyl elongation by blue light in cucumber (Cucumis sativus L.) was studied to examine possible hydraulic and wall changes responsible for diminished growth. Cell-sap osmotic pressure, measured by vaporpressure osmometry, was not decreased by blue light; turgor pressure, measured by the pressureprobe technique, remained constant during the growth inhibition; and stem hydraulic conductance, measured by dynamic and static methods, was likewise unaffected by blue light. Wall yielding properties were assessed by the pressure-block technique for in-vivo stress relaxation. Blue light reduced the initial rate of relaxation by 77%, but had little effect on the final amount of relaxation. The results demonstrate that blue irradiation acts to decrease the wall yielding coefficient, but not the yield threshold. Stress-strain (Instron) analysis showed that irradiation of the seedlings had little effect on the mechanical extensibilities of the isolated wall. The results indicate that blue light can reduce cell-wall loosening without affecting bulk viscoelastic properties, and indicate a chemorheological mechanism of cell-wall expansion.Abbreviations and symbols BL blue light - wall yield coefficient - Y wall yield threshold - P turgor pressure - L hydraulic conductance - g radial water-potential gradient supporting cell expansion - osmotic pressure - P_i initial chamber pressure needed to stop growth - P_f final chamber pressure needed to stop growth     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.