The apical nutations of pea seedlings in relationship to growth regulators

The apical hook curvatures of pea seedlings are associated with a higher level of endogenous auxin on the lower (ventral) side of the curvature in comparison with the upper (dorsal) side. For this reason this curvature may be reversed by applying IAA to the dorsal side of the hook. On the other hand the analyses did not reveal any differences in the content of endogenous gibberellin between the dorsal and ventral side of the hook. Endogenous gibberellin if applied either to the dorsal or to the ventral side of the hook curvature closes the hook. Exogenous cytokinin benzyladenine closes the curvature only if applied to the ventral side of the hook, but it opens it if applied to the dorsal one.     

An analysis of the changes in growth rate occurring during the initial stages of geocurvature in shoots

Abstract A detailed study of the changes in growth rate on the two sides of a shoot prior to, and following geostimulation has been undertaken. Data from etiolated Zea seedlings, and cucumber hypocotyls, and from light grown sunflower hypocotyls are presented. In all cases, the differential growth which brings about geocurvature begins simultaneously along the length of the organ. This is contrary to previous reports in the literature which suggested that curvature begins first at the tip of an organ and progresses basipetally. The feature which is common to all species investigated is that growth of the upper side of the shoot ceases following geostimulation. In some cases there is also a marked acceleration in growth rate on the lower side of the shoot (sunflower) but other species show no such acceleration of growth (cucumber). It has been assumed for many years that the major factor causing upward curvature was an acceleration of growth on the lower side of the organ. The data presented here show that the cessation of growth on the upper side is a major, and in some cases the only factor bringing about geocurvature. The data are discussed in relation to the mechanisms which might control geotropic curvature.     

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.     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

Growth Distribution during Phototropism of Arabidopsis thaliana Seedlings

The elongation rates of two opposite sides of hypocotyls of Arabidopsis thaliana seedlings were measured during phototropism by using an infrared imaging system. In first positive phototropism, second positive phototropism, and red light-enhanced first positive phototropism, curvature toward the light source was the result of an increase in the rate of elongation of the shaded side and a decrease in the rate of elongation of the lighted side of the seedlings. The phase of straightening that followed maximum curvature resulted from a decrease in the elongation rate of the shaded side and an increase in the elongation rate of the lighted side. These data for the three types of blue light-induced phototropism tested in this study and for the phase of straightening are all clearly consistent with the growth rate changes predicted by the Cholodny-Went theory.     

Phototropism in Higher Plants — Controversies and Caveats1

Recent literature on light-induced changes in the distribution of growth and of endogenous growth regulators in the development of phototropic curvature is reviewed. It is concluded that in a great many cases the Cholodny-Went hypothesis is sufficient to account for the growth changes bringing about curvature, though in certain dicotyledonous seedlings differential effects of light on the synthesis of growth inhibitors across the tissue from the lighted to the shaded side are implicated. The problems in the interpretation of results from experiments in which more than one photoresponse may be simultaneously induced are discussed and methods of circumventing them considered. Action spectroscopy suggests that dicotyledonous seedlings may have the same phototropic photoreceptor as monocotyledonous seedlings.     

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.     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

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.     

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.     

A gravitropic stimulation-induced growth inhibitor, β-(isoxazolin-5-on-2yl)-alanine,is a possible mediator of negative gravitropic bending of epicotyls in etiolated <Emphasis Type="Italic">Pisum sativum</Emphasis> seedlings

Negative gravitropic bending and its possible mediator in etiolated Alaska pea seedlings were intensively studied in comparison with seedlings of an agravitropic mutant, ageotropum. When 3.5-day-old etiolated Alaska seedlings were horizontally placed, the growth suppression at the upper side of the epicotyls began 10 min after the onset of the gravitropic stimulation, whereas the growth acceleration at the lower side began at 30 min, resulting in negative gravitropic bending. In contrast, no gravitropic bending was observed in the etiolated ageotropum seedlings, for which the epicotyls show an automorphogenesis-like growth. Strenuous efforts to identify a possible mediator that induces the gravitropic bending resulted in successfully identifying β-(isoxazolin-5-on-2yl)-alanine (βIA). The unilateral application of βIA to the etiolated Alaska epicotyls substantially induced epicotyl bending toward the application site, indicating that βIA could act as a growth inhibitor. Analyses of the distribution of βIA in the upper and lower flanks of the etiolated Alaska epicotyls revealed that its content rapidly increased twice in the upper flanks compared with that in the lower ones in response to gravitropic stimulation, whereas its content in the lower flanks was almost equal to that in the vertical control. In etiolated ageotropum epicotyls, an almost equal amount of βIA was distributed in the upper and lower flanks of epicotyls. These results suggest that a gravitropic stimulation increases βIA in the upper flank, resulting in the negative gravitropic bending of epicotyls via the suppression of the growth rate at the upper side of epicotyls in the etiolated Alaska pea seedlings.     

Rapid, bilateral changes in growth rate and curvature during gravitropism of cucumber hypocotyls: implications for mechanism of growth control

Abstract. The growth response of etiolated cucumber ( Cucumis sativus L.) hypocotyls to gravitropic stimulation was examined by means of time-lapse photography and high-resolution analysis of surface expansion and curvature. In comparison with video analysis, the technique described here has five- to 20-fold better resolution; moreover, the mathematical fitting method (cubic splines) allows direct estimation of local and integrated curvature. After switching seedlings from a vertical to horizontal position, both upper and lower surfaces of the stem reacted after a lag of about 11 min with a two- to three-fold increase in surface expansion rate on the lower side and a cessation of expansion, or slight compression, on the upper surface. This growth asymmetry was initiated simultancously along the length of the hypocotyl, on both upper and lower surfaces, and did not migrate basipetally from the apex. Later stages in the gravitropic response involved a complex reversal of the growth asymmetry, with the net result being a basipetal migration of the curved region. This secondary growth reversal may reflect oscillatory and or self-regulatory behaviour of growing cells. With some qualifications, the kinetics and pattern of growth response are consistent with a mechanism involving hormone redistribution, although they do not prove such a mechanism. The growth kinetics require a growth mechanism which can be stimulated by two-to three-fold or completely inhibited within a few minutes.     

Which cells respond to gravitropic stimulus in the lentil root?

When roots of lentil ( Lens culinaris L., cv. Large blonde) were placed in horizontal position for 2 h, their upper side elongated faster than their lower side, and also faster than vertical controls. The length of the cortical cells was greater in the upper half than in the lower half of roots which had been horizontally stimulated for 2 h. The zone of curvature extended from the distal part of the meristem to the proximal part of the cell elongation zone. The curvature in the meristem was due to early differentiation of the cells of its upper part. In the proximal part of the cell elongation zone, bending took place due to inhibition of cell growth in the lower half of the root. The results obtained are in agreement with the hypothesis of lateral transport of an inhibitor in gravistimulated roots. This inhibitor should be present in greater amounts in the lower side of the stimulated root and in lower amounts in its upper side than in the vertical controls.     

Auxin-mediated response of cucumber seedlings to gravity]

Gravity regulates peg formation because cucumber seedlings grown in a horizontal position develop a peg on the lower side of the transition zone (TR zone) but not on the upper side. Studies on peg formation have suggested the regulation of peg formation by gravity as follows. Cucumber seedlings potentially develop a peg on both the lower and upper sides of the TR zone. The development of the peg on upper side of the TR zone is suppressed in response to gravity. A phytohormone, auxin, induces peg formation. Upon gravistimulation the auxin concentration on the upper side of the TR zone is reduced to a level below the threshold value necessary for peg formation. The unequally distributed auxin across TR zone is caused by a change in accumulation of auxin influx carrier (CsAUX1) protein and auxin efflux carrier (CsPIN1) protein in response to gravity. In addition, TR zone before peg initiation expresses both CsARF2 (putative activator of auxin response factor) and CsIAA1 (putative repressor of auxin-inducible gene expression), by which TR zone could respond the auxin gradient regulated by gravity.     

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.     

Gravitropic Responses of Partially Decapitated Corn Coleoptiles with and without Applied [C]Indoleacetic Acid

The curvature of corn seedling (Zea mays L. Mo17 × B73) coleoptiles which had been half-decapitated and supplied with [¹⁴C]indoleacetic acid (IAA) (3.2 micromolar, 51 milliCuries per millimole) was determined during a 3-hour period of gravitational stimulation. Curvature of such half-decapitated coleoptiles was found to be similar in rate and extent to that of intact coleoptiles responding to gravity. Gravitational stimulation was accomplished by reorienting seedlings to a horizontal position, either up or down with respect to the removed half of the coleoptile tips.

The first set of experiments involved placing aluminum foil barriers along one of the two cut surfaces to restrict the movement of IAA into tissues. The initiation and extent of curvature of these half-decapitated coleoptiles was dependent upon the orientation of the removed half-tip and the accompanying barrier. The distribution of radioactivity from [¹⁴C] IAA after 3 hours indicated that the specific lateral movement of label was also dependent upon orientation of the removed half-tip of the coleoptile. A specific movement to the lower side of approximately 14% of the total recovered radioactivity was found in coleoptiles in which the [¹⁴C]IAA was supplied across a transverse cut surface. In contrast, specific movement of only 4% was found for application across a longitudinal cut surface.

A second series of experiments was conducted using 1.0 and 3.2 micromolar [¹⁴C]IAA (51 milliCuries per millimole) supplied to half-decapitated coleoptiles without inserted barriers. The 3.2 micromolar concentration adequately replaced the removed coleoptile half-tips in terms of straight growth, but it did not result in as much curvature as shown by coleoptiles of intact seedlings. The 1 micromolar concentration was not adequate to replace the removed half-tip in straight growth, but resulted in gravitropic curvature nearly as great as that produced by the higher concentration.

The data presented here suggest that strong auxin gradients are not produced in response to gravity stimulation based on the recovered radioactivity from [¹⁴C]IAA. However, it is evident that auxin is required for the development of normal gravitropic responses. It is possible, therefore, that an important early role of this movement is not to cause a large stimulation of growth on the lower side but to decrease growth on the upper side of a gravitropically responding coleoptile.

     

Gravity induces fast electrical field change in soybean hypocotyls

Abstract. Measurement of the electrical field along soybean hypocotyls shows the development of a positive electrical potential in the lower side approximately 1 min after horizontal placement. The time is as fast or faster than the geotropic presentation time of soybean seedlings. The maximum positive electrical field potential is produced in a zone 1–2 cm below the hook, which is the region showing the geotropic curvature.     

Phototropism in Hypocotyls of Radish: IV. Flank Growth and Lateral Distribution of cis- and trans-Raphanusanins in the First Positive Phototropic Curvature

The first positive phototropic curvature induced by a pulse of unilateral white irradiation (0.1 watt per square meter, 30 seconds) of etiolated and de-etiolated Sakurajima radish (Raphanus sativus var hortensis f. gigantissimus Makino) hypocotyls was analyzed in terms of differential growth and growth inhibitor contents of the hypocotyls. In both etiolated and de-etiolated hypocotyls, the growth rates at the lighted sides were suppressed whereas those at the shaded ones showed no change. De-etiolation treatment induced a larger difference between the growth rates at the lighted and shaded sides of the hypocotyls, resulting in a larger curvature of de-etiolated seedlings than of etiolated ones. The contents of growth inhibitors, cis- and trans-raphanusanins, increased in the lighted but not in the shaded halves of the hypocotyls of etiolated seedlings. In de-etiolated seedlings, the two inhibitors increased due to the de-etiolation treatment. When de-etiolated seedlings were exposed to a pulse of unilateral irradiation the level of the two inhibitors remained high along the lighted side for 1 h following the light pulse, whereas at the shaded side the contents of the inhibitors abruptly decreased upon transfer to the dark, the difference between their amounts in the lighted and shaded sides being larger than in etiolated seedlings. Another growth inhibitor, raphanusamide, did not respond to the phototropic stimulus, although its amounts increased by the de-etiolation treatment. These data suggest that cis- and trans-raphanusanins are involved in the first positive phototropic response of radish hypocotyls, and that de-etiolation magnifies the phototropic response through induction of a larger lateral gradient of the raphanusanins in the hypocotyls by the phototropic stimulus.     

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.     

Ethylene evolution changes in the stems of<Emphasis Type="Italic"> Metasequoia glyptostroboides</Emphasis> and<Emphasis Type="Italic"> Aesculus turbinata</Emphasis> seedlings in relation to gravity-induced reaction wood formation

Two-year-old Metasequoia glyptostroboides and 3-month-old Aesculus turbinata seedlings were tilted at a 45° angle to induce compression wood formation on the lower side of the former species and tension wood on the upper side of the latter. Two weeks later, the seedlings were tilted in an opposite direction at 45° so that the upper and lower sides changed to each other. This reverse tilting was kept for 7 weeks for M. glyptostroboides and 6 weeks for A. turbinata. The seedlings were sampled and analyzed at intervals throughout each experimental period so that an ethylene evolution kinetic was monitored. Ethylene evolution from the cambial region of the upper and lower sides of tilted stems was measured separately by gas chromatography with a flame ionization detector. Xylem production expressed as wood area during each experimental period was microscopically determined. In both tilting and reverse tilting periods, the rates of ethylene evolution from the lower side of M. glyptostroboides and the upper side of A. turbinata, where xylem production was accelerated and compression or tension wood formation was induced, had increased to high levels, whereas those from the opposite sides had either remained low (in tilting period) or rapidly recovered to low levels (in reverse tilting period). The cambial activity quantified by wood formation, including reaction wood, in both species showed the same tendency as ethylene evolution. The stem side with vigorous ethylene evolution, xylem development and reaction wood formation reversed with the reversal of tilting orientation. The roles of accelerated ethylene evolution in reaction wood formation in the tilted seedlings of gymnosperm and angiosperm trees are compared and discussed.This work was presented at the 5th Pacific Region Wood Anatomy Conference, Yogyakarta, Indonesia, 9–14 September 2002