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)     

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.     

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m(-2) s(-1)). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0-3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0-3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0-2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.     

Changes in cellular osmotic potential and mechanical properties of cell walls during light-induced inhibition of cell elongation in maize coleoptiles

The growth rate of maize ( Zea mays L. cv. Cross Bantam T51) coleoptiles in the dark was highest at the basal zone and decreased towards the tip. Growth was strongly inhibited by white fluorescent light (5 W m⁻²), especially in the basal zone of coleoptiles. Light irradiation caused an increase in the values of stress-relaxation parameters, the minimum stress-relaxation time and the relaxation rate and a decrease in the extensibility (strain/stress) of the cell walls at all zones. In addition, during growth, the accumulation of osmotic solutes was strongly inhibited by white light irradiation, resulting in an increased osmotic potential. The influences of white light on the mechanical properties of the cell wall and the osmotic potential of the tissue sap were most prominent in the basal zone. Significant correlations were observed between the increment of coleoptile length and the mechanical properties of the cell walls or the osmotic potential of the tissue sap and osmotic solutes content. Furthermore, light inhibited the outward bending of split coleoptile segments. These facts suggest that white light inhibits elongation of maize coleoptiles by modifying both the mechanical properties of the cell walls and cellular osmotic potential, which control the rate of water uptake.     

Auxin induces exocytosis of acid phosphatase in coleoptiles from Zea Mays

Auxin-stimulated elongation growth of maize coleoptiles has been suggested to be associated with enhanced exocytotic activity. However, the problem in plants is one of finding a soluble parameter, which can be used as a direct measure of exocytosis (H. D. Blackbourn and N. H. Battey [1993]. Physiol. Plant. 89: 27–32). In yeast, acid phosphatase (EC 3.1.3.2) is used as a marker for secretory activity (E. Harsay and A. Bretscher [1995]. J. Cell Biol. 131: 297–310). Therefore, extracellular acid phosphatase activities in maize tissues were investigated. Coleoptile (7.36 nkat mg^-1) and mesocotyl (8.9) showed higher specific extracellular acid phosphatase activities than primary leaf (6.0), root (4.9) and root tip (2.7). In coleoptiles extracellular acid phosphatase activity was 6.7% of total homogenate activity (mesocotyls 10.6%). Auxin (30 μM IAA) increased the extracellular acid phosphatase activity of coleoptiles (146% of control). This effect was tissue-specific; extracellular acid phosphatase activity of mesocotyls was not enhanced by IAA. The stimulating effect of auxin on extracellular acid phosphatase activity in coleoptiles was reversed by the protonophore nigericin (0.3 μM). Furthermore, localization of an acid phosphatase activity in Golgi vesicles was shown by co-migration of the Golgi marker latent IDPase (EC 3.6.1.6) and acid phosphatase activity (65% of total microsomal activity) on isopycnic continuous sucrose density gradients. Tonoplast-enriched membrane frctions (24% of microsomal acid phosphatase) and plasma membrane-enriched fractions (11%) contained lower amounts of acid phosphatase. The data presented suggest that acid phosphatase activity is a useful marker for hormone-induced secretory activity in plant cells.     

Growth distribution during first positive phototropic curvature of maize coleoptiles*

Abastract Measurements of growth increments on the shaded and the irradiated sides of phototropically stimulated maize (Zea mays L.) coleoptiles, obtained over the entire fluence range of the first positive curvature, indicate that the curvature is induced by growth stimulation on the shaded side and compensating inhibition on the irradiated side (length increments on the coleoptile flanks were determined 100 min after 30 s phototropic induction with blue light). At high fluences of blue light, overall stimulation of growth takes place, but this tendency is largely eliminated when only the tip of the coleoptile is irradiated. Time courses for growth increments obtained for the maximum first positive response show that the growth stimulation on the shaded side and the growth inhibition on the irradiated side commence almost simultaneously 20-30 min after the phototropic induction. The growth on the irradiated side almost ceases, but the growth rate on the shaded side is doubled, relative to the control rate. The onset of differential growth migrates basipetally from the tip at a velocity similar to that for polar auxin transport. The first positive phototropic response of the coleoptile is concluded to be the consequence of lateral redistribution of growth, which is not necessarily accompanied by changes in the net growth. The results are consonant with the Cholodny-Went theory of tropisms, in which lateral redistribution of auxin is considered to be the cause of tropic responses.     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in maize root segments

In apical or subapical root segments of maize ( Zea mays L. cv. Dekalb XL640), 10⁻⁷–10⁻⁵ M 24-epibrassinolide (BR), a physiologically active synthetic epimer of the pollen hormone brassinolide, induces a significant stimulation of root growth, associated with an increase of acid secretion. The increase in acid secretion is enhanced by the presence of K⁺ in the medium and is accompanied by an early, significant hyperpolarization of the transmembrane electric potential (PD), which is completely suppressed by the addition of the protonophore uncoupler FCCP. Similar effects of BR have earlier been reported for shoots, and also for IAA in shoots. Contrariwise, 10⁻⁸–10⁻⁷ M IAA inhibits acid secretion and depolarizes the PD in the maize root segments. This suggests different pathways for the action of the two different hormones on the proton pump.     

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.     

Le transport basipète de l'AIA-5-3H dans le coléoptile de blé

Basipetal transport of [5-³H] IAA in the wheat coleoptile
³H-IAA was applied to the tips of intact wheat coleoptiles ( Triticum sativum L., var. Capitole) for 30, 60 or 120 min. The quantity of label per segment (1.5 mm), determined by liquid scintillation counting, was a decreasing exponential function of distance from the source of radioactivity. Chromatographic analysis showed that ³H-IAA was transported more rapidly than its tritiated metabolites. Antoradiographs of semi-thin sections were performed after application of ³H-IAA during 2 h and after treatment by DCC [1-(3-dimethyl-aminopropyl)-3-ethycarbodiimide hydrochloride]. The quantity of label per tissue and per cell was determined at 1.5, 3.0 and 4.5 mm from the tip. Tritiated IAA was estimated to account for 70 to 100% of the label, depending on the distance from the tip. The amount of auxin transported basipetally was greatest in the parenchyma, with lesser amounts transported in the inner and outer epidermis. Tissues of the vascular bundles did not contain more than 10% of total radioactivity of the coleoptile sections. The rate of auxin transport was greater in xylem and parenchyma than in epidermis and bundle sheath. Movement was very slow in procambium and phloem. In parenchyma and epidermis the quantity of label per cell as a function of distance from the tip agreed with the model of exponential regression. These results support the theory of polar diffusion of auxin.     

Rapid growth responses of dark-grown wheat seedlings to red-light irradiation. II. Kinetic studies on the growth of different coleoptile zones

Continuous recordings of the effect of red light on the over-all and zonal growth responses were made on intact, dark-grown wheat ( Triticum aestivum L., cv. Hatri) seedlings selected 70 or 90 h after sowing. The over-all growth response of intact coleoptiles induced by bilateral continuous red light (660 nm, 17 W m⁻²) was complex and resulted from the overlapping of different zonal growth responses. During a 5 h investigation period, these responses can be divided into two phases. The first phase (short-term response) was a transient growth inhibition. After a lag period of ca 15 min, the rate of extension decelerated to a minimum value at ca 60 min, after which an acceleration was seen. This response was qualitatively the same in all coleoptile zones investigated (tip, subapical zone, base) and independent of coleoptile age. The second phase (delayed response) became measurable between 1.8 and 3 h after onset of red light irradiation and exhibited zonal-specific growth promotion or inhibition, dependent on the coleoptile age. A persistent growth promotion was observed only in the tip region of coleoptiles selected 70 h after sowing and became detectable about 3 h after the onset of red light.     

Effects of γ-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles

The effects of γ-irradiation on elongation and the level of indole-3-acetic acid (IAA) of maize (Zea mays) coleoptiles were investigated. When 3-day-old seedlings of maize were exposed to γ-radiation lower than 1 kGy, a temporal retardation of coleoptile elongation was induced. This retardation was at least partly ascribed to a temporal decrease in the amount of free IAA in coleoptile tips on the basis of the following facts: (1) the reactivity to IAA of the elongating coleoptile cells was not altered by irradiation; (2) endogenous IAA level in the tip of irradiated coleoptiles was at first unchanged, but then declined before returning to nearly the same level as that of the non-irradiated control; and (3) the amount of IAA that diffused from coleoptile tip sections showed a similar pattern to that of endogenous IAA. The rate of conversion between free and conjugated IAA was not significantly affected by irradiation. These results suggest that a temporal inhibition of maize coleoptile elongation induced by γ-irradiation can be ascribed to the reduction of endogenous IAA level in the coleoptile tip, and this may originate from the modulation in the rate of IAA biosynthesis or catabolism.     

Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles

When maize coleoptiles were unilaterally exposed to red light (7.9 μmol m⁻²s⁻¹ for 5 min), 3 h after treatment IAA levels in coleoptiles decreased in all regions, from top to basal, with levels about 60% of dark controls. Localized irradiation in the 5 mm top zone was sufficient to cause the same extent of IAA reduction in the tips to that in the tips of whole irradiated shoots. When coleoptiles were treated with N-1-naphthylphthalamic acid (NPA), an accumulation of IAA in the tip and a decrease of diffusible IAA from tips were simultaneously detected. IAA accumulation in red-light treated coleoptiles by NPA was much lower than that of dark controls. NPA treatment did not affect the content of conjugated IAA in either dark or light treated coleoptile tips. When ¹³C₁₁ ¹⁵N₂-tryptophan (Trp) was applied to the top of coleoptiles, substantial amounts of stable isotope were incorporated into free IAA in dark and red-light treated coleoptile tips. The ratio of incorporation was slightly lower in red-light treated coleoptile tips than that in dark controls. The label could not be detected in conjugated IAA. The rate of basipetal transport of IAA was about 10 mm h⁻¹ and the velocity was not affected by red light. These results strongly suggest that red light does not affect the rates of conversion of free IAA to the conjugate form or of the basipetal transport, but just reduces the IAA level in the tips, probably inhibited by IAA biosynthesis from Trp in this region.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

The Movement and Physiological Effect of Indoleacetic Acid Following Point Applications to Root Tips of Zea mays

Pinpoint applications of labeled and non-labeled indoleacetic acid (IAA) on resin beads were made, without injury, to vertical roots of intact seedlings of Zea mays. Points of application were at the extreme tip of the root, 0.5, 2 and 5 mm from the root tip. The movement of label and bending of the roots was recorded. Radiolabel was found to move basipetally from the extreme tip and 0.5 mm applications to a similar extent, reaching 8 mm from the tip. The level of label in the growing zone after 4 h was 10% of that found in the extreme tip. Movement from 2 and 5 mm applications was equal in both directions. Higher amounts of non-labeled IAA caused bending towards the point of application if applied at 0.5 or 2 mm but not at 5 mm from the tip. It is proposed that any endogenous IAA in the root cap could move to the growing zone and cause a unilateral inhibition of growth, provided that it was in the same transport pool as the exogenously applied IAA.     

Distribution of Endogenous Indole-3-acetic Acid and Growth Inhibitor(s) in Phototropically Responding Oat Coleoptiles

The relationship between the flank growth of oat (Avena sativaL. cv. Victory) coleoptiles and the distribution of endogenousindole-3-acetic acid (IAA) and growth inhibitor(s) in the coleoptileswas studied for the second positive phototropic curvature inducedby a continuous unilateral illumination with white light (0.1W.m^–2). The phototropic curvature was caused by growthinhibition at the lighted side and growth promotion at the shadedside. Using electron capture detection gas chromatography, weanalyzed the distribution of endogenous IAA in phototropicallyresponding oat coleoptiles and found that the IAA was evenlydistributed over the lighted and shaded sides during the phototropicresponse; there was also no detectable difference in the amountsof IAA between phototropically stimulated and non-irradiatedcoleoptiles. By contrast, oat coleoptile straight-growth testresults showed that the amount of unknown acidic growth inhibitor(s),different from abscisic acid, increased in the lighted halfof the coleoptiles and decreased in the shaded half, as comparedto the amount in the non-irradiated half. These data suggestthat the phototropic curvature of oat coleoptile is inducedby a difference in lateral flank growth through a lateral gradientof endogenous growth inhibitor(s) rather than of IAA. (Received February 10, 1988; Accepted July 29, 1988)     

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.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.     

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.     

PHYTOCHROME ACTION IN ORYZA SATIVA L. III. THE SEPARATION OF PHOTOPERCEPTIVE SITE AND GROWING ZONE IN COLEOPTILES, AND AUXIN TRANSPORT AS EFFECTOR SYSTEM

• 1 In 4-day-old etiolated rice seedlings, 3 mm of the coleoptile tip did mainly perceive the photostimulus to cause the phytochrome-dependent inhibition of coleoptile elongation. At this age, cell elongation occurred most in the middle portion of coleoptiles in the dark, and was reversibly controlled by a brief exposure of the tip to red and far-red light. Thus, the photoperceptive site was evidently separated from the growing zone in intact rice coleoptiles.
• 2 The red-light-induced inhibition of coleoptile elongation was nullified by the removal of tip followed by the exogenous application of IAA. The sensitivity of thus treated coleoptiles to IAA was gradually lost during intervening darkness between the irradiation and the decapitation, and a 50% loss was obtained at ca. 6th hour at 26°C.
• 3 Polar auxin transport from coleoptile tips was remarkably prevented at the period between, at least, 2nd and 4th hour after red irradiation, and it recovered to the level of dark control by the 6th hour. Far-red light given immediately after red irradiation reversed the yield of diffusible auxin up to that of far-red control.

     

Effect of zeatin on the growth and indolyl-3-acetic acid and abscisic acid levels in maize roots

Elongation, indolyl-3-acetic acid (IAA) and abscisic acid (ABA) levels, – gas chromatography-mass spectrometry quantification –, in the elongating zone were analysed for maize ( Zea mays L., Cv. LG11) roots immersed in buffer solution with or without zeatin (Z). The effect of Z depends on the initial extension rate of roots. The slower growing roots are more strongly inhibited by Z (10⁻⁷−10₋₅ M ) and they show a greater increase in IAA and ABA content. When compared to the rapidly growing roots, the larger reactivity of the 'slow'ones cannot be attributed to a higher Z uptake as shown when using [¹⁴C]-Z. It is suggested that Z could regulate root elongation by acting on the IAA and/or ABA level. The comparative action of these two hormones is discussed.