Gravitropic responses of the Avena coleoptile in space and on clinostats. I. Gravitropic response thresholds

We conducted a series of gravitropic experiments on Avena coleoptiles in the weightlessness environment of Spacelab. The purpose was to test the threshold stimulus, reciprocity rule and autotropic reactions to a range of g-force stimulations of different intensities and durations The tests avoided the potentially complicating effects of earth's gravity and the interference from clinostat ambiguities. Using slow-speed centrifuges, coleoptiles received transversal accelerations in the hypogravity range between 0.1 and 1.0 g over periods that ranged from 2 to 130 min. All responses that occurred in weightlessness were compared to clinostal experiments on earth using the same apparatus.
Characteristic gravitropistic response patterns of Avena were not substantially different from those observed in ground-based experiments. Gravitropic presentation times were extrapolated. The threshold at 1.0 g was less than 1 min (shortest stimulation time 2 min), in agreement with values obtained on the ground. The least stimulus tested, 0.1 g for 130 min, produced a significant response. Therefore the absolute threshold for a gravitropic response is less than 0.1 g.     

Gravitropic responses of the Avena coleoptile in space and on clinostats. IV. The clinostat as a substitute for space experiments

Gravitropic responses of dark grown oat coleoptiles were measured in weightlessness and under clinorotation on earth. The tests in microgravity were conducted in Spacelab during the IML-1 mission and those on clinostats were conducted in laboratories on earth. The same apparatus was used for both kinds of tests. In both cases autotropism and gravitropic responsiveness were determined. This allowed a quantitative comparison between the plants' responses after receiving the same tropistic stimulations either in weightlessness or on clinostats.
Autotropism was observed with oat coleoptiles responding in weightlessness but it did not occur on clinostats. Gravitropic responsiveness was measured as the ratio between the incremental bending response (degrees curvature) and the corresponding incremental g-dose (stimulus intensity times duration for which it was applied). Plants were tested at either of two stages of coleoptile development (i.e. different coleoptile lengths). From a total of six different kinds of critical comparisons that could be made from our tests that provided data for clinorotated vs weightless plants, three showed no significant difference between responses in simulated vs authentic weightlessness. Three other comparisons showed highly significant differences. Therefore, the validity of clinorotation as a general substitute for space flight was not supported by these results.     

Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles

We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature.     

Effects of submergence on development and gravitropism in the coleoptile of Oryza sativa L.

Caryopses of rice (Oryza sativa L. cv. Sasanishiki) were germinated in air or under water. In submerged seedlings a twofold increase in coleoptile growth rate and an inhibition of root growth was observed. The amount of starch in the amyloplasts of submerged coleoptiles was substantially reduced compared to the air-grown control plants and plastids had a proplastidic character. During the rapid elongation of coleoptiles under water, the osmotic concentration of the press sap remained constant, whereas in air-grown coleoptiles a decrease was measured. Determination of curvature of gravistimulated air-grown and submerged shoots was carried out by placing the coleoptiles horizontally in air of 98% relative humidity. Air-grown coleoptiles reached a vertical orientation within 5 h after onset of gravistimulation. In coleoptiles germinated under water the first signs of consistent negative gravitropic bending occurred after 4–5 h and curvature was complete after 24 h. During the first 5 h of gravistimulation the water-grown coleoptiles grew at an average rate of 0.39 mm·h^–1, whereas in air-grown coleoptiles a rate of 0.27 mm·h^–1 was measured. Concomitant with the delayed onset of gravitropic bending of the water-grown coleoptiles, a change in plastid ultrastructure and an increase in starch content was observed. We conclude that the gravitropic responsiveness of the rice coleoptile depends on the presence of starch-filled amyloplasts.We wish to thank H.-J. Ensikat for technical assistance with the scanning electron microscopy. Supported by the Bundesminister für Forschung und Technologie and the Deutsche Forschungsgemeinschaft.     

Intracellular localization of the active process in polar transport of auxin

Summary The cytoplasm of maize coleoptile cells was displaced to either the apical or basal ends of the cells by centrifuging (1750xg for 10 min) segments in which protoplasmic streaming had been stopped by pretreatment with cytochalasin B. Centrifugation toward the base of the segment promotes the subsequent basipetal transport of indole-3-acetic acid, whereas apical centrifugation dramatically inhibits this transport. Apical centrifugation neither promotes acropetal transport nor reverses the polarity of auxin transport. Experiments in which the amyloplasts were separated from the bulk of the cytoplasm indicate that the basipetal transport is independent of both the position and pressure exerted by the amyloplasts but is strongly dependent on the amount of cytoplasm at the basal end of the cells. These effects of centrifugation on auxin transport lead to the conclusion that the metabolic component of the transport is a polar secretion of auxin localized in the basal plasma membrane of each cell.     

Plasmodesmata, Tropisms, and Auxin Transport

Attempts were made to disrupt the plasmodesmata between oatcoleoptile cells (Avena saliva L. cv. Victory) by severe plasmolysis.Coleoptiles, allowed to regain turgor after plasmolysis, wereable to execute geotropic and phototropic curvatures and segmentswould grow in response to applied auxin. In coleoptiles similarlytreated, studies with [¹⁴C]IAA have shown that longitudinal,basipetal transport of auxin still takes place and, as in controls,IAA is preferentially redistributed laterally within coleoptilesorientated horizontally. Physical continuity of the symplast of oat coleoptile cellsmay not always be disrupted by severe plasmolysis. Nevertheless,functional continuity appears to be interrupted. Despite this,all the processes involved in the execution of tropistic curvaturesremain intact, including transport of hormones. Plasmodesmatalcontinuity between oat coleoptile cells appears not to be anecessary requirement for auxin transport.     

Can Lateral Redistribution of Auxin Account for Phototropism of Maize Coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.     

Effect of gravity and centrifugal acceleration on auxin transport in corn coleoptiles

Summary Inversion of corn coleoptile sections resulted in a 10–20% inhibition of basipetal transport of 3-indoleacetic acid (IAA) and a more pronounced inhibition (20–50%) of the transport of 1-naphthaleneacetic acid (NAA).The effect of inversion on basipetal NAA transport was compared in wild-type corn and in the amylomaize mutant which contains smaller and slower sedimenting amyloplasts: the gravity induced inhibition was higher in the wild type coleoptiles (27% versus 9%).In wild type the inhibitory effect on basipetal NAA transport appeared within less than 30 min after inversion; then the effect remained relatively constant over at least 2 hr of transport. When the sections were returned to the upright position the transport rate increased, reaching the level of upright controls within 30 min.An effect of gravity on lateral transport of NAA was also demonstrated and shown to be expressed within 10 min after placing the tissue horizontally.When basipetal transport was tested in the direction of gravity and/or centrifugal acceleration, auxin movement incrased with increasing acceleration. Transport against centrifugal acceleration (10 x g) was less than transport of control sections (inverted at 1 x g).The results agree with the hypothesis that starch statoliths act by a pressure mechanism on the membrane transport system of auxin.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Auxin Transport in Zea mays Coleoptiles II. Influence of Light on the Transport of Indoleacetic Acid-2-C

The effect of bilateral irradiation with white light (1000 Meter Candle Sec) on the basipetal transport of auxin has been investigated. Illumination of either the intact shoot or the excised coleoptile tip of the Zea seedling, decreased the amount of diffusible auxin obtained from the tip, and decreased Avena curvature response to unilaterally applied indoleacetic acid. Irradiation of the intact Zea seedling did not affect the absorption of ¹⁴C-labeled indoleacetic acid from an agar block subsequently placed on the decapitated coleoptile. However, light caused a significant decrease in the amount of labeled auxin basipetally transported, without affecting materially the velocity of that transport. These and other observations are interpreted as support for the hypothesis that the primary hormonal phenomenon in first-positive phototropism is a light-induced impairment in the basipetal transport of auxin.     

Effects of Morphactin on Indol-3yl-acetic Acid Transport, Growth, and Geotropic Response in Cereal Coleoptiles

The effects of the morphactin 2-ehloro-9-hydroxyfluorene-9-carboxylicacid methyl ester [CFM] on growth, geotropic curvature and transportand metabolism of indol-3yl-acetic acid [IAA-5-³H] in the coleoptilesof Zea mays and A vena saliva have been investigated. A strongcorrelation has been found to exist between the inhibition ofthe geotropic response and the inhibition of auxin transport.CFM supplied at concentrations sufficient to abolish auxin transporthas been shown to promote the elongation of Zea, but not ofAvena, coleoptile segments. CFM does not change the patternof metabolism of IAA in Zea coleoptile segments. In these segmentsIAA is metabolized when its concentration is high, but the radioactivitytransported basipetally, or laterally in geotropically stimulatedcoleoptiles, is virtually confined to the IAA molecule. Radioactivityexported into the basal receiver blocks is wholly confined toIAA. It is concluded that CFM inhibits the geotropic responsein coleoptiles by suppression of the longitudinal and lateralauxin transport mechanisms. The growth-promoting propertiesof this substance cannot be linked with its effects on eitherauxin metabolism or transport.     

Cholodny–Went revisited: a role for jasmonate in gravitropism of rice coleoptiles

Gravitropism is explained by the Cholodny–Went hypothesis: the basipetal flow of auxin is diverted laterally. The resulting lateral auxin gradient triggers asymmetric growth. However, the Cholodny–Went hypothesis has been questioned repeatedly because the internal auxin gradient is too small to account for the observed growth asymmetry. Therefore, an additional gradient in indolyl-3-acetic acid (IAA) sensitivity has been suggested (Brauner and Hager in Planta 51:115–147, 1958). We challenged the Cholodny–Went hypothesis for gravitropism of rice coleoptiles (Oryza sativa L.) and found it to be essentially true. However, we observed, additionally, that the two halves of gravitropically stimulated coleoptiles responded differentially to the same amount of exogenous auxin: the auxin response is reduced in the upper flank but normal in the lower flank. This indicates that the auxin-gradient is amplified by a gradient of auxin responsiveness. Hormone contents were measured across the coleoptile by a GC-MS/MS technique and a gradient of jasmonate was detected opposing the auxin gradient. Furthermore, the total content of jasmonate increased during the gravitropic response. Jasmonate gradient and increase persist even when the lateral IAA gradient is inhibited by 1-N-naphtylphtalamic acid. Flooding with jasmonate delays the onset of gravitropic bending. Moreover, a jasmonate-deficient rice mutant bends more slowly and later than the wild type. We discuss a role of jasmonate as modulator of auxin responsiveness in gravitropism.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Polarity and rate of transport of cyclic adenosine 3,5'-monophosphate in the coleoptile

Transport of tritiated cyclic AMP in the coleoptile of oats (Avena sativa) and corn (Zea mays) is polar, with basipetal to acropetal ratios of 4.0 and 3.2, respectively. The rate of transport is approximately that of indoleacetic acid. The linear velocity of transport, however, is at least five times that of auxin. A loss in transport polarity of the nucleotide occurs in subapical tissues within several hours after decapitation of the coleoptile, accompanied by a decrease in transport rate. The loss in polarity is not reversed by exogenous auxin, but the reduction in transport is. Auxin also inhibits the uptake of cyclic AMP. Exogenous cyclic AMP is metabolized rapidly by coleoptile tissues. If cyclic AMP does have a cellular function in the coleoptile, its transport behavior is compatible with that of a hormone.     

Movement of Indoleacetic Acid in Coleoptiles of Avena sativa L. II. Suspension of Polarity by Total Inhibition of the Basipetal Transport

Acropetal and basipetal movement of indole-3-acetic acid through coleoptiles of Avena sativa L. was studied. Sections 10-mm long were supplied with either apical or basal sources containing C(14) carboxyl-labeled indoleacetic acid (10(-5)m). Anaerobic conditions inhibit metabolically dependent movement (transport) thus reducing basipetal but not acropetal movement. Total inhibition of basipetal transport abolishes the polarity of auxin uptake and movement. The nonpolar movement that remains in anaerobic sections is free diffusion with an average diffusion coefficient of approximately 1 x 10(-4) mm(2) per second. During an 8-hour diffusion, at least the first millimeter of the section comes to equilibrium at approximately the same concentration as the donor.Acropetal movement is probably by diffusion and is accompanied by an aerobic immobilization of indoleacetic acid that increases more than proportionally to concentration. Anaerobic conditions totally prevent this immobilization and reduce acropetal uptake but not the amount of indoleacetic acid moving into the upper parts of the section; there is, therefore, no evidence for acropetal transport.Polarity of auxin movement in aerobic coleoptile sections is achieved by strict basipetal transport of auxin. The basipetal transport may intensify the polarity by recycling auxin that is moving acropetally.     

Auxin-induced elongation of short maize coleoptile segments is supported by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one

Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 μM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much smaller in segments shorter than 2–3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially purified methanol extract of maize coleoptiles supported long-term, auxin-controlled elongation. The active material was also found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is not the general auxin cofactor but may specifically “spare” the co-auxin in maize. Received: 27 June 2000 / Accepted: 16 October 2000     

Different forms of phototropic response induced by microbeam irradiation of discrete regions of an organ

Abstract Continuous unilateral microbeam (1 mm) irradiation of coleoptiles of Avena saliva L. produces growth responses in non-irradiated regions of the organ. Irradiation of the apex is followed by differential growth which commences in the apical zone, but then moves in a basipetal direction and results in positive curvature. Irradiation of the base also results in differential growth which commences at the apex and moves basipetally. However, the growth differential in this case results in negative curvature. The results of basal irradiation suggest the likely occurrence of acropetal transmission of the phototropic signal. The rate of movement of this signal is faster than the documented rate of auxin transport.     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

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.

     

Some aspects of geotropism in coleoptiles

Summary Auxin transport was studied in coleoptile sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 min of horizontal exposure. After about 15 min this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptile. Upon return of the coleoptile to a vertical position, this asymmetry disappeared within 30 min.Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropically induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. In all pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 (67) had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23%).Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles.Further results, confirming the increased effect of centrifugal acceleration greater than 1xg on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.with participation by Charles steele and Vicky fan