Coleoptile Sections Research Articles

Physiological Mechanism of the Auxin-Induced Increase in Light Sensitivity of Phytochrome-Mediated Growth Responses in Avena Coleoptile Sections

The physiology of the auxin-induced 10,000-fold increase in light sensitivity of a phytochrome-mediated growth response (Shinkle and Briggs, 1984 Proc Natl Acad Sci USA 81: 3742-3746) has been characterized in subapical coleoptile sections from dark-grown oat (Avena sativa L. cv Lodi) seedlings. Six micromolar indole-3-acetic acid (IAA) must be present for 1 hour before to 2 hour after irradiation in order to confer maximal sensitivity to light. The direct effect of IAA on growth can be separated from its effect on light sensitivity. Several classes of synthetic auxins will substitute for IAA in inducing an increase in sensitivity to light, as will both the phytotoxin fusicoccin and treatment of sections with pH 4.5 buffer. The increase in sensitivity to light induced by 6 micromolar IAA is completely inhibited by buffering the sections at pH 5.9 with 30 millimolar 2-(N-morpholino)ethanesulfonic acid. These findings suggest that the capacity to respond to very low fluences of light is regulated by extracellular pH.Between 10 and 15 millimolar K(+) will inhibit the induction of the increased sensitivity to light, independent of the mechanism of induction. The effect of K(+) appears to be specific to the process by which the sections respond to very low levels of light.

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside.

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). II. Localization of a putative auxin receptor.

With the aid of affinity chromatography on auxin-binding protein-Sepharose (ABP-Sepharose) monospecific IgGanti-ABP from rabbit antisera were isolated as judged by immuno-double diffusion test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this IgGanti-ABP the ABP is localized within the outer epidermal cells of coleoptiles using indirect immunofluorescence labeling. Auxin-induced growth of coleoptile segments can be inhibited by IgGanti-ABP, and the auxin response of split coleoptile sections is also strongly reduced by IgGanti-ABP. The ABP, therefore, is referred to as an auxin receptor. This auxin receptor is localized at the plasmalemma of the outer epidermal cells of the coleoptile.

Tunicamycin‐induced growth and inhibition of glucosamine incorporation into cell walls of rice coleoptiles

Tunicamycin, 0.25 to 2.5 μM, promotes elongation of rice coleoptile sections after a 2 h lag. Tunicamycin decreased the minimum stress‐relaxation time of the cell wall, T0; and the wall loosening is recognized as the cause of this growth promotion. Bacitracin did not have significant effects on growth or T0 except for inhibition of elongation at high concentration. Coleoptile sections were incubated with [14C]‐glucosamine, and the synthetic pathway of the hexosamine‐containing cell wall component was examined by a pulse‐chase experiment. This component seems to be synthesized in the particulate fraction and secreted mainly into the hemicellulose I fraction. Tunicamycin strongly inhibited glucosamine incorporation into the particulate fraction and stopped the labeling of the cell wall. At 2.5 μM, tunicamycin had no effect on incorporation of mannose, leucine or proline. These results suggest that the hexosamine‐containing wall component is a kind of asparagine‐linked glycoprotein, and that this component plays a principal role in formation of the cell wall network and in growth regulation.

Rapid reduction by IAA of malondialdehyde levels in avena coleoptiles, a possible effect on lipid peroxidation

Treatment of Avena coleoptile sections with IAA results in a rapid decrease in the level of lipid peroxidation (LP), as measured by the thiobarbituric acid reaction for malondialdehyde (MDA). The response is specific for active auxins, is nearly saturated by 10(-6) M IAA and occurs even when turgor is reduced by 0.1 M mannitol. About half the reduction in MDA occurs within 1 min after addition of IAA to intact tissues; addition of IAA directly to homogenates, however, has no effect. Homogenates prepared from auxin-pretreated tissues, but not control tissues, continue to produce MDA over at least a six-hour period. This effect of auxin on LP is one of the most rapid biochemical responses to auxin known, and suggests that LP might alter the properties of membranes and thus influence cell enlargement.

Cell wall pH and auxin transport velocity.

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.

Indole-3-acetic acid sensitization of phytochrome-controlled growth of coleoptile sections

Addition of 6 muM indole-3-acetic acid (IAA) to incubation buffer increases the sensitivity of coleoptile sections cut from dark-grown Avena sativa L. cv. Lodi to red light by a factor of 10,000, relative to the response in the absence of added IAA, without changing the maximum amount of light-induced growth. From 0.03 to 4 muM IAA sections show at least a 100-fold increase in sensitivity to red light relative to the response in the absence of added IAA. In this IAA concentration range, the light-induced increase in elongation shows two phases of response to red-light fluence, which are separated by a plateau. The biphasic fluence-response curve is also characteristic of the red-light-induced stimulation of coleoptile growth in intact dark-grown seedlings. The effect of IAA on the sensitivity of the phytochrome-mediated growth response appears to be on some step in the transduction of the phytochrome signal, rather than on the growth response itself.

Auxin Concentration/Growth Relationship for Avena Coleoptile Sections from Seedlings Grown in Complete Darkness

A biphasic auxin dose-response curve has been obtained for indole-acetic acid (IAA)-stimulated growth of subapical sections of coleoptiles from totally dark-grown oats (Avena sativa L. cv Lodi). The curve for growth at 6 h is composed of a log-linear phase and a modified bell-shaped phase separated by a plateau. The curve is log-linear from 0.003 to 0.4 micromolar IAA when sections are incubated in pH 5.9 buffer. The plateau of IAA concentration-neutral growth is seen from 0.4 to 4.0 micromolar IAA. Further increase in growth occurs from 4.0 to 10 micromolar IAA. Changing the pH of the buffer from 5.9 to 5.5 or 6.2 changes the shape of the curve, shifting the plateau to lower IAA concentration, or abolishing it, respectively. The synthetic auxin 2,4-dichlorophenoxyacetic acid also shows a biphasic dose-response curve, but the synthetic auxin 1-naphthalene acetic acid does not. The plateau is not affected by the auxin-transport inhibitor 2,3,5-triiodobenzoic acid. The plateau is eliminated by taking sections from coleoptiles grown under continuous dim red light. We advance a model to account for these results based on two modes of auxin uptake into the cell: carrier-mediated uptake and uptake via chemiosmotic diffusion.

Relation of gibberellic acid-induced growth of wheat coleoptiles with indole acetic acid

Summary Gibberellic acid (GA3) induced elongation of wheat coleoptile sections and its relation with endogenous indole acetic acid was studied. Growth promotion by GA was reduced to half when oxidation of endogenous IAA was prevented by pretreatment of the coleoptiles with EDTA or a mixture of chlorogenic acid and 2,2′-dipyridyl. The effect of GA was reduced to ¼ when the endogenous methyleneoxindole (MeOx), an oxidation product of IAA, was made unavailable to GA3 during its action. External application of hydroxymethyloxindole (HMO), earliest oxidation product of IAA, helped GA to regain its full activity even when oxidation of endogenous IAA was inhibited. It is concluded that endogenous IAA, more precisely, its oxidation products, were necessary for GA in exhibiting its growth promoting activity on wheat coleoptile sections.

Rapid growth responses of dwarf corn coleoptile sections to picloram

The initial and transient growth responses of coleoptile sections of Dwarf-1 corn ( Zea mays L.) to picloram (4-amino-3,5,6-trichloropicolonic acid) were studied and compared to those produced after IAA (indole-3-acetic acid) treatments. Growth was measured every 36 sec with an angular displacement transducer. Picloram-mediated and also IAA-mediated growth recorded approximately 10 min of lag period before any growth response was elicited. The rate of picloram-mediated growth then increased rapidly, reaching a maximum between 25 and 40 min after treatment. This was followed by a second maximum approximately 80 min after treatment; then the growth rate slowly decreased to a constant level. The shape of the rate curve of picloram-mediated growth was essentially the same as that obtained after IAA treatment. Analysis of growth kinetics suggests that modes of growth stimulating action of picloram may not involve IAA metabolism inplants, but suggests that the compound is itself an auxin.

Suppression of auxin stimulated growth of barley coleoptile sections by endosulfan

Endosulfan, a cyclic sulphurous acid ester commonly used as a broad spectrum insecticide, suppressed the elongation of barley coleoptiles. Indoleacetic acid at optimum concentration overcame the inhibition of growth of coleoptiles treated with 10 ppm endosulfan. However, perfusion of the coleoptile sections with endosulfan and subsequent treatment with indoleacetic acid could not stimulate cell elongation to the extent observed in the control

Changes in isoperoxidases induced by hydroxyphenylacetic acids

The effects ofo-, m- andp-hydroxyphenylacetic acids on total peroxidase activity and isoperoxidase patterns of wheat coleoptile sections were studied.o-andm-HOPA reduced the total peroxidase activity whilep-HOPA increased it. Electrophoresis in polyacrylamide gel revealed thato- andm-HOPA diminish the intensity of some anodic and cathodic bands, but thatp-HOPA increases anodic and reduces cathodic isoperoxidases. These results may explain the apparently paradoxical positive effects ofp-HOPA on both growth and total peroxidase activity.

Structure-activity differences between indoleacetic acid auxins on pea and wheat

A series of halogenated indoleacetic acids was assessed for auxin activity on pea stem and wheat coleoptile sections. Activity differences between the two species were found. These are discussed, in terms of differences in receptor models for pea and wheat, with the models differing in the areas covered by the 6- and 7-substituted compounds.

Effects of Chlorpromazine on Gravitropism in Avena Coleoptiles

Chlorpromazine (CPZ), an inhibitor of the calcium-activated form of calmodulin, is readily taken up by the roots of intact oat seedlings but poorly translocated from the roots to the coleoptile of these plants. However, plants repeatedly rotated through solutions containing low concentrations of CPZ (10~8-10~5 m) are infiltrated, and under these conditions, CPZ significantly inhibits the negative gravitropic response of the coleoptile without retarding elongation growth. This effect is observable in 4 decapitated* (apical 1-2 mm removed) coleoptile sections and in intact whole coleoptiles. If exogenous auxin is supplied to the decapitated sections, both their growth rates and gravitropic responsiveness are increased and, under these conditions, CPZ can reduce the gravitropic curvature without reducing the overall growth rate. These results are discussed in relation to the possible role of calmodulin-dependent calcium-ion pumps in gravitropism.

Barium effects inPhaseolus aureus, Cephalandra indica,Canna indica, Beta vulgaris, Triticum aestivum andLactuca sativa

Barium chloride at 0.1 mM concentration inhibited elongation of mungbean (Phaseolus aureus) roots more strongly than elongation of hypocotyls. Root growth was completely inhibited at 80 mM and there was no germination at 100 mM. Respiration rates which were directly proportional to seedling vigour declined at varying degrees at the growth-inhibitory concentrations of BaCl2. Barium retarded senescence in isolated leaf discs ofCephalandra indica by maintaining the chlorophyll level in darkness. The carotenes were similarly protected from degradation by Ba in darkness whereas xanthophylls were retained both in darkness and light. Ba also protected anthocyanin pigments in the staminode discs ofCanna indica flowers and reduced to a variable extent the leakage of betacyanin pigment fromBeta vulgaris root discs caused by various membrane active chemicals. Applied either alone or in combination with IAA, Ba inhibited cell enlargement in wheat (Triticum aestivum) coleoptile sections. Barium-induced inhibition of lettuce (Lactuca sativa) hypocotyl elongation was largely overcome by GA3.

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.

Biological Properties of d-Amino Acid Conjugates of 2,4-D

Some d-amino acid (glutamic acid, valine, or leucine) conjugates of 2,4-dichlorophenoxyacetic acid (2,4-D) at 10(-5) molar, stimulated elongation of Avena sativa L. var Mariner coleoptile sections and growth of soybean (Glycine max. L. var Amsoy) tissue as much as did the l-amino acid conjugates at 10(-6) molar. The d-methionine conjugate did not stimulate growth of soybean root callus tissue but did stimulate Avena elongation. The d-aspartic acid conjugate did not stimulate elongation of Avena coleoptiles but did stimulate growth of root callus tissue.Pretreatment of root callus tissue with 100 micromolar 2,4-D-(d) -or (l) glutamic acid for 18 hours prior to incubation for 6 hours in [1-(14)C]2,4-D resulted in a greater uptake of [1-(14)C]2,4-D than did pretreatment with 2,4-d. Qualitatively the metabolites were similar in all preincubation treatments. Preincubation with 2,4-D-(d)-glutamic acid also resulted in larger free [(14)C]2,4-D pool and a significant increase in water-soluble conjugates when compared with 2,4-D, or 2,4-D-(l)-glutamic acid preincubations.

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [(3)H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5-5 μM IAA is included, the pulse achieves its maximum velocity of about 2 cm h(-1). At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [(3)H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A(-)) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [(3)H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A(-). The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A(-) component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.

Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis

According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate "wall loosening" enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.Excised coleoptile sections accumulated radioactive glucose, arabinose, and xylose supplied in an incubation medium, and the radioactivity from these sugars was incorporated into polysaccharides. At least 50% of the radioactivity from glucose accumulated in the soluble neutral sugar fraction regardless of external concentrations. The distribution of radioactivity from xylose into all subcellular fractions was similar to that from glucose, indicating that xylose was converted to glucose before being used by the coleoptile. IAA increased the incorporation of glucose into cell wall polysaccharide and neutral sugar pools when the exogenous concentration was higher than 1 millimolar.Over 80% of the radioactivity from arabinose accumulated by the coleoptile sections was incorporated into soluble and noncellulosic polymers; IAA induced an increase in the incorporation of arabinose into noncellulosic polymers by 22%. Accumulation of radioactivity from arabinose into polysaccharide was enhanced by IAA at concentrations of exogenous arabinose up to 33 millimolar.IAA promoted the incorporation of both arabinose and glucose into cell wall polysaccharides even when elongation was inhibited by CaCl(2), indicating that the influence of IAA was not a consequence of the growth response.

Osmoregulation in the Avena Coleoptile

Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations.