Gravitropism and Phototropism of Maize Coleoptiles: Evaluation of the Cholodny-Went Theory Through Effects of Auxin Application and Decapitation

It was investigated whether or not gravitropism and phototropismof maize (Zea mays L.) coleoptiles behave as predicted by theCholodny-Went theory in response to auxin application, decapitationand combinations of these treatments. Gravitropism was inducedat an angle of 30° from the vertical, and phototropism,by a pulse of unilateral blue light. Either tropism of the coleoptilewas inhibited by IAA, applied as a ring of IAA-lanolin pasteto its sub-apical part, and by decapitation. The dose-responsecurves for the effects of applied IAA on tropisms and growthof intact coleoptiles as well as the time courses of tropismsinduced in decapitated coleoptiles could be explained by thethree conclusions in the literature: (1) the tip of the coleoptileis the site of auxin production, (2) lateral translocation ofauxin in gravitropism occurs along the length of the coleoptile,and (3) lateral translocation of auxin in phototropism occursin the coleoptile tip. By examining the effects of decapitationmade at different distances from the top and of IAA appliedto the cut surface of decapitated coleoptiles, it was indicatedthat auxin is produced in the apical 1 mm zone of an intactcoleoptile and that lateral auxin translocation for phototropismtakes place in an apical part that somewhat exceeds the zoneof auxin production. (Received October 14, 1994; Accepted December 26, 1994)     

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.     

Phototropism: mechanisms and ecological implications

Abstract. Phototropism in seed plants, either etiolated or de-etiolated, is mediated by unidentified photoreceptor(s) sensitive to blue and near-UV regions of the light spectrum. Green plants may have an additional phototropic system sensitive to red light. Fluence-response studies of the blue light-sensitive phototropism, initially made on oat coleoptiles, have indicated the occurrence of multiple response types. Of those, two are found to be general: the first pulse-induced positive phototropism (fPIPP), or the so-called first positive curvature, and the time-dependent phototropism (TDP) or the second positive curvature. The fPIPP, elicited by a pulse stimulus shorter than a few minutes, is characterized by a bell-shaped fluence-response curve and the validity of reciprocity. The TDP, elicited by prolonged irradiation, is characterized by its dependence on the exposure time and the invalidity of reciprocity. Studies made on these two response types have revealed the following: (1) plants acquire directional light information for phototropism by sensing internal light gradients created by light scattering and absorption; (2) phototropism results from redistribution of growth, i.e. inhibition on the irradiated side and compensating stimulation on the shaded side; (3) lateral movement of growth regulators, the principle of the Cholodny-Went theory, can account for the growth redistribution, and auxin is clearly the mediating regulator in maize coleoptiles. This review further describes some mechanistic implications of fPIPP. Experimental results indicate that (1) fPIPP is mediated by a single step of photoreaction, (2) the responsiveness, reflected in the height of the fluenceresponse curve, is reduced by pre-irradiation with blue light and recovers gradually afterward, and (3) the light sensitivity, reflected in the position of the fluence-response curve along the log fluence axis, is also reduced by the pre-irradiation and recovers gradually. Analyses of these results, based on kinetic models, suggest that the bell-shaped fluence-response curve is caused by the difference in the amounts of a photoproduct between irradiated and shaded sides, and that fPIPP represents a mechanism of TDP. It is also indicated that phytochrome in the red-absorbing form exerts two separate effects on phototropism: reduction of the light sensitivity and enhancement of the responsiveness. Along with the discussion of the mechanisms of phototropism, their ecological implications are considered.     

Phytochrome action in Oryza sativa L.

Summary The mechanical properties of the cell wall were measured in coleoptiles of totally etiolated rice seedlings. Coleoptiles were either decapitated or briefly exposed to red (R) and/or far-red (FR) light. The elastic and plastic extensibilities of the cell wall changed with age (length) of the coleoptiles. Decapitation and exposure to R induced changes in these properties, and the time-courses were similar. Following decapitation or R irradiation, the plastic extensibility of the cell wall decreased more conspicuously than elastic extensibility. Exogenous application of auxin immediately following decapitation alleviated the effect of removal of the tip. FR irradiation reduced both kinds of extensibilities, but its effect was much less than that of R, and it reversed the R-induced effect to the level of tissue treated with FR only. In repeated R-FR treatments, the decrease of elastic extensibility by R and its reversal by FR could be repeated, but the effect of a second irradiation with R after FR on plastic extensibility was not as apparent as that of the first. Reduction of cell-wall extensibility of etiolated rice coleoptiles caused by R light appeared, at least partly, to be due to a reduced auxin supply in the elongating region from the tip, similar to that caused by decapitation.     

Phytochrome is required for the occurrence of time-dependent phototropism in maize coleoptiles

Time-dependent phototropism (TDP), sometimes called second positive curvature, occurs when the duration of phototropic stimulation with blue light (B) exceeds a few minutes. TDP was characterized in maize (Zea mays L.) coleoptiles raised under continuous red light (R). Subsequently, coleoptiles adapted to darkness were used to investigate the effect of R on TDP. It was found that TDP, which is induced in R-grown coleoptiles, does not occur in dark-adapted coleoptiles and that dark-adapted coleoptiles begin to show TDP after treatment with R. The TDP responsiveness became maximal 1-2 h after treatment with a R pulse and decreased during the next few hours. At least 10 min was required after a short pulse of R before the coleoptile began to respond to B for the induction of TDP. The effect of R in establishing the TDP responsiveness was totally suppressed by a pulse of far-red light given immediately after an inductive pulse of R. It is concluded that the mechanism of TDP requires for its establishment a R signal perceived by phytochrome. The TDP of R-grown and R-pretreated coleoptiles showed relationships to stimulation times and fluence rates that are similar to those reported for oat coleoptiles, except that TDP of maize showed a sharp increase in its magnitude within a narrow range of stimulation times as short as 5-10 min.     

Polarity induction versus phototropism in maize: Auxin cannot replace blue light

In a previous study (Nick and Schäfer 1991, Planta 185, 415–424), unilateral blue light had been shown, in maize coleoptiles, to induce phototropism and a stable transverse polarity, which became detectable as stable curvature if counteracting gravitropic stimulation was removed by rotation on a horizontal clinostat. This response was accompanied by a reorientation of cortical microtubules in the outer epidermis (Nick et al. 1990, Planta 181, 162–168). In the present study, this stable transverse polarity is shown to be correlated with stability of microtubule orientation against blue light and changes of auxin content. The role of auxin in this stabilisation was assessed. Although auxin can induce reorientation of microtubules it fails to induce the stabilisation of microtubule orientation induced by blue light. This was even true for gradients of auxin able to induce a bending response similar to that ellicited by phototropic stimulation. Experiments involving partial irradiation demonstrated different perception sites for phototropism and polarity induction. Phototropism starts from the very coleoptile tip and involves transmission of a signal (auxin) towards the subapical elongation zone. In contrast, polarity induction requires local action of blue light in the elongation zone itself. This blue-light response is independent of auxin.This work was supported by the Deutsche Forschungsgemeinschaft and two grants of the Studienstiftung des Deutschen Volkes and the Human Frontier Science Program Organization to P.N.     

The Rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin

We isolated a mutant, named coleoptile phototropism1 (cpt1), from gamma-ray-mutagenized japonica-type rice (Oryza sativa). This mutant showed no coleoptile phototropism and severely reduced root phototropism after continuous stimulation. A map-based cloning strategy and transgenic complementation test were applied to demonstrate that a NPH3-like gene deleted in the mutant corresponds to CPT1. Phylogenetic analysis of putative CPT1 homologs of rice and related proteins indicated that CPT1 has an orthologous relationship with Arabidopsis thaliana NPH3. These results, along with those for Arabidopsis, demonstrate that NPH3/CPT1 is a key signal transduction component of higher plant phototropism. In an extended study with the cpt1 mutant, it was found that phototropic differential growth is accompanied by a CPT1-independent inhibition of net growth. Kinetic investigation further indicated that a small phototropism occurs in cpt1 coleoptiles. This response, induced only transiently, was thought to be caused by the CPT1-independent growth inhibition. The 3H-indole-3-acetic acid applied to the coleoptile tip was asymmetrically distributed between the two sides of phototropically responding coleoptiles. However, no asymmetry was induced in cpt1 coleoptiles, indicating that lateral translocation of auxin occurs downstream of CPT1. It is concluded that the CPT1-dependent major phototropism of coleoptiles is achieved by lateral auxin translocation and subsequent growth redistribution.     

玉米胚芽鞘向光性运动的一些特性

利用云母片分隔、ＨＰＬＣ分析等方法研究了玉米胚芽鞘向光性运动的特性。云母片阻隔生长素的移动后并不能阻止胚芽鞘的向光性变弯曲。     

Physiology of Movements in the Stems of Seedling Pisum sativum L. cv Alaska : III. Phototropism in Relation to Gravitropism, Nutation, and Growth

Phototropic response in etiolated pea (Pisum sativum L. cv Alaska) seedlings is poor. However, the curvature induced by unilateral blue light can be hastened and increased in magnitude by a previously administered red light pulse followed by several hours of darkness. Phytochrome is involved in the red light effect. Phototropic response was almost completely inhibited by removal of the apical bud and hook, but it was restored if exogenous indole-3-acetic acid was applied apically to the cut stump. Therefore, the stem contains both the phototropic photoreceptor and response mechanism. Perception of gravity and gravitropic response were also localized in the stem, but gravitropism was scarcely inhibited by decapitation. It was also observed that the kinetics and curvature pattern of gravitropism differed greatly from those of phototropism. Like phototropism, stem nutation required auxin and was promoted by red light. Unlike phototropism, photoenhanced nutational curvature required the apical hook and was propagated as a wave down the stem. Naphthylphthalamic acid inhibited, in order of decreasing effect, nutation, phototropism/gravitropism, and growth. Phototropism, gravitropism, and nutation appear to represent distinct forms of stem movement with fundamental differences in the mechanisms of curvature development.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Auxin dynamics after decapitation are not correlated with the initial growth of axillary buds

One of the first and most enduring roles identified for the plant hormone auxin is the mediation of apical dominance. Many reports have claimed that reduced stem indole-3-acetic acid (IAA) levels and/or reduced basipetal IAA transport directly or indirectly initiate bud growth in decapitated plants. We have tested whether auxin inhibits the initial stage of bud release, or subsequent stages, in garden pea (Pisum sativum) by providing a rigorous examination of the dynamics of auxin level, auxin transport, and axillary bud growth. We demonstrate that after decapitation, initial bud growth occurs prior to changes in IAA level or transport in surrounding stem tissue and is not prevented by an acropetal supply of exogenous auxin. We also show that auxin transport inhibitors cause a similar auxin depletion as decapitation, but do not stimulate bud growth within our experimental time-frame. These results indicate that decapitation may trigger initial bud growth via an auxin-independent mechanism. We propose that auxin operates after this initial stage, mediating apical dominance via autoregulation of buds that are already in transition toward sustained growth.     

IAA-Induced Growth Responses of Decapitated Corn Seedlings: Indications of Two Apparent Adaptations with a Possible Role in Gravitropism

The vertical growth responses of corn seedlings (Zea mays L. Mo17 × B73) were determined over an 8-hour period. When seedlings were decapitated 3 millimeters from the coleoptile's tip and supplied with indole-3-acetic acid (IAA) in 1.5% agar blocks, the response was dependent both on time and IAA concentration. The dose-response curves changed in shape and magnitude depending on the total time of IAA application. High concentrations (>3.2 × 10⁻⁶ molar) initially produced high relative growth rates that decreased back to the intact rate (0.03 millimeter per hour per millimeter) after 3 hours. Low concentrations (<1.0 × 10⁻⁶ molar), or agar blocks without IAA, resulted in a rapid decrease from the intact rate to a level that stabilized at 0.01 millimeter per hour per millimeter until the growth rate began to recover after 3 to 4 hours. Intermediate concentrations produced responses similar to that of the intact organ, though some features of these responses were unique.

The coleoptile curvature in response to gravity depended upon whether the coleoptiles were intact, decapitated, or decapitated and supplied with IAA. Coleoptiles decapitated and not supplied wth IAA showed little or no curvature for 3 hours after decapitation. By this time an adaptation, evoked by the low IAA level, had developed and the coleoptiles began to curve steadily. When 1.0 or 3.2 × 10⁻⁶ molar IAA was supplied, curvature was initiated within the first 30 minutes and reached a maximum rate before decreasing and stopping after 3 to 4 hours. The sequence of events in response to these concentrations was similar to the intact sequence but the curvature rate was reduced to one-third to one-half. A model for the autotropic response involving an auxin concentration-dependent, growth-modulating mechanism capable of two modes of adaptation is described.

     

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.     

Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana,axr2

Gravitropism and phototropism of the primary inflorescence stems were examined in a dominant Aux/IAA mutant of Arabidopsis, axr2/iaa7, which did not display either tropism in hypocotyls. axr2-1 stems completely lacked gravitropism in the dark but slowly regained it in light condition. Though wild-type stems showed positive phototropism, axr2 stems displayed negative phototropism with essentially the same light fluence-response curve as the wild type (WT). Application of 1-naphthaleneacetic acid-containing lanolin to the stem tips enhanced the positive phototropism of WT, and reduced the negative phototropism of axr2. Decapitation of stems caused a small negative phototropism in WT, but did not affect the negative phototropism of axr2. p-glycoprotein 1 (pgp1) pgp19 double mutants showed no phototropism, while decapitated double mutants exhibited negative phototropism. Expression of auxin-responsive IAA14/SLR, IAA19/MSG2 and SAUR50 genes was reduced in axr2 and pgp1 pgp19 stems relative to that of WT. These suggest that the phototropic response of stem is proportional to the auxin supply from the shoot apex, and that negative phototropism may be a basal response to unilateral blue-light irradiation when the levels of auxin or auxin signaling are reduced to the minimal level in the primary stems. In contrast, all of these treatments reduced or did not affect gravitropism in wild-type or axr2 stems. Tropic responses of the transgenic lines that expressed axr2-1 protein by the endodermis-specific promoter suggest that AXR2-dependent auxin response in the endodermis plays a more crucial role in gravitropism than in phototropism in stems but no significant roles in either tropism in hypocotyls.     

植物光敏素在玉米胚芽鞘不同类型向光性反应中的作用模式

先前的研究考察了红光对玉米（Zea mays L.cv.Royaldent Hit85)胚芽鞘向光性反应的影响,本研究进一步分析了红光对蓝光照射时间依赖型向光性（TDP）影响的光具－反应曲线,发现该反应不像红光对脉冲蓝光诱导的向光性的影响那样属于超低光量反应,而是一种低光量反应,且之后的脉冲远红光可以逆转红光对TDP影响的效果。但远红光预处理延长后,逆转了的TDP反应性可以得到恢复。不仅如此,暗适应胚芽鞘接受不同时间的单独远红光预处理后可同样获得与预处理光量成比例的TDP反应性,表明暗适应胚芽鞘在接受远红光顶处理后亦可建立起长时间向光性蓝光照射的反应能力。远红光对TDP反应性影响的光量－反应曲线分析揭示,该远红光影响依赖于照射时间并要求高光量。鉴于高光量范围内上述远红影响不符合所谓反比定律,远红光对TDP反应性的影响很可能属一高辐照反应,根据上述研究发现探讨了植物光敏素作用模式与不同光性反应信号转导途径之间可能存在的相互关系。     

Modes of Action of Phytochromes in Establishing DifferentPhototropic Responsiveness of Maize Coleoptiles

Previous studies have examined the effects of red light (R) on phototropism of maize ( Zea mays L. cv. Royaldent Hit 85) coleoptiles. The R effect on time-dependent phototropism (TDP) was further studied by characterizing its fluence-response relationship. The results showed the R effect was a low-fluence-response, unlike those on pulse-induced phototropisms that show a very-low-fluence-response mode. A subsequent pulse of far-red light (FR) could reverse the R effect. TDP responsiveness, however, recovered as the following FR was extended. The FR-dependent increase in TDP responsiveness was obtained even coleoptiles were pretreated only with FR. It suggested that TDP responsiveness could also be established in response to a FR signal. The fluence-response relationship for the effect of FR was then investigated. The effect of FR depended on the time of irradiation and required high photon fluences. Because reciprocity was invalid at the higher fluence range, the effect of FR would be a high-irradiance-response mode. Relation between phytochrome action modes and possible multiple pathways for phototropic signal transduction was analyzed based on the experiment results.     

Influence of the Shoot Tip and Leaves on Circumnutation in Green Pea Seedlings

Portions of the shoot system from young light-grown pea (Pisum sativum L.) seedlings were excised and circumnutation studied using time-lapse cinematography. Removal of the youngest leaf or shoot tip as well as ringing the stem with 20 mM triiodobenzoic acid severely restricted circumnutation. Treating the stump of the excised leaf with lanolin containing 10^?4 M indole-3-acetic acid or replacing the leaf with an artificial aluminum leaf both partially restored circumnutation. When the leaf was replaced with both auxin and an artificial leaf circumnutation continued at approximately the rate of the intact plant. This graphically shows the involvement of both auxin and gravitropism in circumnutation.     

Root-shoot Hormone Relations I. The Importance of an Aerated Root System in the Regulation of Growth Hormone Levels in the Shoot of Helianthus annuus

Using mature, vegetative, sunflower (Helianthus annuus) plants,a study was made of the interactions of flooding of the rootsystem with several other simultaneous treatments. Leaf epinastywas induced in intact plants by flooding, but this effect ofwaterlogging of the root system was removed when the shoot wasdecapitated. The addition of indole-3-acetic acid (IAA) in lanolinto the cut surface of decapitated plants restored the epinasty-inducingeffect of flooding. This is taken as evidence that floodingbrings about an increased auxin content of the shoot. In unfloodedplants decapitation of the shoot resulted in a hyponastic movementby the leaves, and applications of IAA to the stem tip antagonizedthis. Girdling of flooded plants resulted in a great reduction ofthe tendency to form adventitious roots in the region belowthe girdle. Thickening and various morphological changes wereproduced in the hypocotyl as a result of waterlogging the rootsystem. The effects of flooding could be simulated in the upperregion of the shoot of unflooded plants by girdling the morebasal region of the stem. These various results are interpreted as indicating that theroot system may normally serve as a centre for the oxidativeinactivation of excess shoot-synthesized auxin, so regulatingshoot auxin level. Certain of the results obtained, however,show that an adequately aerated root system supplies some growthpromoting,and hyponasty-inducing, principle to the shoot. The effectsof this hypothetical principle were found to be similar to thoseof gibberellic acid. It is envisaged, therefore, that an earlyeffect of flooding on plants is to raise shoot auxin levels(either by preventing entry of auxin from the stem, or by inhibitingthe oxidative catabolism of shoot auxin in the roots), and alsoto stop the synthesis of a non-auxin shoot growth-hormone inthe roots. The induction of wilting of the shoot by floodingwas found to be correlated with low root-temperatures.     

Effects of red light on the fluence–response relationship for pulse-induced phototropism of maize coleoptiles

Dark-adapted coleoptiles of maize (Zea mays L.) were treated with red light (3min at 10.5 μmol m^?2S^?1) and were Stimulated, after a dark interval, with a pulse of unilateral blue light to induce phototropism. Phototropic fluence-response curves were obtained in this way for different dark intervals. It was confirmed that the bell-shaped fluence-response curve for the first pulse-induced positive phototropism (FPIPP) shifts to higher fluences following the red-light treatment, the maximal shift being achieved at a dark interval of 2h. We found, however, that the two arms of the Fluence-response curve do not shift synchronously. The shift of the descending arm to higher fluences began at 15 min. The ascending arm showed a slight shift to lower fluences before a greater shift to higher flucnces. the change of the shift direction occurring at 30–40min. Accordingly, the fluence-response curve obtained for a 30 min dark interval was comparatively wide. Although dark-adapted coleoptiles showed only fPIPP, another bell-shaped fluence-response curve, representing the second pulse-induced positive phototropism (sPIPP), appeared gradually after the red-light treatment. These changes of the phototropic fluence– respnse curve following exposure to red light are likely to have adaptive values because they favour phototropism under brighter light.     

Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.