Bound indoleacetic Acid in Avena coleoptiles

When C¹⁴ carboxyl indoleacetic acid (IAA) is transported through Avena coleoptile sections a fraction of the activity becomes bound. The nature of this bound IAA has been investigated. Upon extraction with solvents and chromatography a substance having the R_F of IAA in 4 solvents was detected. No evidence could be found for the formation of indoleacetyl conjugates. In pea stem sections subjected to a similar experimental regime good evidence was obtained for the occurrence of conjugates. When IAA was supplied exogenously to coleoptile sections floating in solutions the occurrence of conjugates was shown to be dependent on the presence of the primary leaf. In its absence no conjugates could be detected.

On grinding coleoptile sections and subsequent centrifugation at 240 × g the radioactivity was found to be in the tissue fraction as opposed to the supernatant. The radioactivity cannot be removed from the tissue by extraction with water, buffer solution or treatment with ribonuclease. It is readily removed by 10% urea, crystalline trypsin and chymotrypsin. It is therefore concluded that IAA becomes bound to a protein. Bound IAA does not appear to be able to cause growth in Avena coleoptile sections.

     

Transport of indoleacetic Acid in intact corn coleoptiles

We have characterized the transport of [³H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration.     

Promotion of indoleacetic Acid oxidase isoenzymes in tobacco callus cultures by indoleacetic Acid

Indoleacetic acid oxidase in tobacco callus cultures (Nicotiana tabacum L., cv. White Gold) was composed of at least two groups of isoenzymes, which were distinctly different in electrophoretic mobilities and in responses to growth substances. Indoleacetic acid had dual effects; at low concentrations it promoted the development of two fast-migrating indoleacetic acid oxidase isoenzymes, but at high concentrations it increased the level of other indoleacetic acid oxidase isoenzymes with low and moderate electrophoretic mobilities. However, indoleacetic acid was not unique in such effects; 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were effective at concentrations lower than that of indoleacetic acid.     

Effects of indoleacetic Acid on dictyosomes of apical and expanding cells of oat coleoptiles

We found that the auxin-induced growth is mediated through the activation of the dictyosomes (collectively, the Golgi apparatus). Incubation of oat (Avena sativa) coleoptile segments in indoleacetic acid-sucrose-phosphate buffer changes significantly the number of dictyosomes in the expanding cells. A further indication of auxin enhancement of dictyosome activity is a decrease in dictyosomal cisternae (flattened membranous sacs) number. This decrease occurred after 6 minutes of incubation in auxin, and then was followed by a reduction in the organelle number per se. These times are in keeping with the rapid action of auxin-induced cell elongaton, and the latent period of geotropism. In the apical cells, the effect of indoleacetic acid is more subtle and complex. The periods of increased dictyosome utilization and of increased dictyosome synthesis in auxin-treated segments alter with those of the control. These observations indicate that dictyosomes not only have a function in cell elongation, but also may participate in processes such as auxin transport and stimuli perception. The expanding cells have five times as many dictyosomes as the cells in the apex. Dictyosome number within a cell appears to be directly proportional to the length of the cell. The fluctuation of dictyosome number and the effect of auxin on the rate of elongation of individual outer epidermis are discussed.     

A red-far red reversible effect on uptake of exogenous indoleacetic Acid in etiolated rice coleoptiles

The uptake and accumulation of exogenous indoleacetic acid-¹⁴C by intact rice coleoptiles were examined. The absorption of exogenous indoleacetic acid was controlled by phytochrome, while the subsequent accumulation of this indoleacetic acid in various portions of the coleoptile was complex, and the effect of red light in this system was small compared to the alteration of the uptake of indoleacetic acid by red light. The absorption of indoleacetic acid exhibited two phases: the first occurring during the first 3-hour portion of the incubation was an inhibition, while the second was a promotive effect at about the 5th hour of incubation. Both of these effects were red, far redreversible, implicating phytochrome in this effect. Neither the destruction nor the immobilization of this exogenous indoleacetic acid apeared to be greatly affected by red light irradiation. The principal interaction between phytochrome and indoleacetic acid appears to occur during the absorption of exogenous indoleacetic acid. This effect may be related to the control by phytochrome of the amount of auxin which diffuses from coleoptile tips.     

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H⁺ excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H⁺ excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H⁺ in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O₂ uptake and before the increased rate of Rb⁺ uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H⁺ excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H⁺ excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H⁺ excretion by IAA and FC.     

Comparison of the effect of indoleacetic Acid and fusicoccin on the breakdown of phosphatidylinositol in maize coleoptiles

The effect of indoleacetic acid (IAA) and fusicoccin (FC) on the breakdown of phosphatidylinositol in maize (Zea mays L.) coleoptiles has been studied. Coleoptiles were able to incorporate [³H] myo-inositol into the phospholipid fraction almost linearly for 8 hours. Thin layer chromatography analysis of total phospholipids showed that [³H]myo-inositol was incorporated only into phosphatidylinositol. Prelabeled coleoptiles treated with IAA showed a loss of the radioactivity incorporated in the phospholipid fraction, whose level decreased by 34% after 1 hour. Treatment with FC, on the contrary, did not modify the content of labelled phosphatidylinositol with respect to the control. The different effects of IAA and FC and a possible mechanism of IAA action on growth are discussed.     

Oxidized oligogalacturonides activate the oxidation of indoleacetic Acid by peroxidase

Partial hydrolysis of polygalacturonic acid with a purified α-1,4-endopolygalacturonase yielded oligogalacturonides and trace amounts of a series of modified oligogalacturonides. Three of the minor products were isolated and identified as oxidized oligogalacturonides possessing termini of galactaric acid. Oxidation of indole-3-acetic acid by peroxidases was activated by oxidized oligogalacturonides but not by normal analogs.     

Changes in indoleacetic Acid oxidase isoenzymes in tobacco tissues after treatment with 2,4-dichlorophenoxyacetic Acid

2,4-Dichlorophenoxyacetic acid had a multiple effect on the development of indoleacetic acid oxidase isoenzymes in tobacco callus tissues (Nicotiana tabacum, cv. White Gold) cultured in vitro, and the development of these isoenzymes was differentially associated with growth promotion or inhibition depending on the concentration of 2,4-dichlorophenoxyacetic acid. At low concentrations (0.1 to 1 μm) it promoted the development of a fast migrating isoenzyme A₅ accompanied by stimulation of a tumor-type growth. At high concentrations (10 to 100 μm), it inhibited the development of the fast migrating isoenzymes but promoted a sharp rise in others with slower electrophoretic mobilities, which was accompanied by growth inhibition. The implications are that 2,4-dichlorophenoxyacetic acid might alter the level of endogenous auxins through its dual effects on the oxidase isoenzyme system.     

The effect of chitooligosaccharides on hydrogen peroxide production and anionic peroxidase activity in wheat coleoptiles

We studied the effects of chitooligosaccharides (ChOS) with a mol wt of 5 kD, the degree of acetylation of 65%, and the concentrations from 0.01 to 100 mg/l on the content of hydrogen peroxide in incubation medium and the activity of anionic peroxidase (pI 3.5) in the segments of wheat (Triticum aestivum) coleoptiles. H₂O₂ production and peroxidase activity were found to be dependent on the ChOS concentration. After 3 h of incubation, the highest H₂O₂ level in medium was observed at 0.01 mg/l ChOS, whereas after 6h, at 1 mg/l. After 3 h of incubation, ChOS suppressed peroxidase activity. After 6 h of incubation, high ChOS concentrations enhanced peroxidase activity. IAA favored H₂O₂ accumulation in medium and suppressed anionic peroxidase. The involvement of ChOS in the control of the level of reactive oxygen species and anionic peroxidase activity in plant cells is suggested.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 238–242.Original Russian Text Copyright © 2005 by Yusupova, Akhmetova, Khairullin, Maksimov.This revised version was published online in April 2005 with a corrected cover date.     

Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Increase in indoleacetic Acid oxidase activity of winter wheat by cold treatment and gibberellic Acid

The activity of indoleacetic acid oxidase increased 10-fold during 40 days of cold treatment of winter wheat seedlings. Puromycin and 6-methyl purine inhibited indoleacetic acid oxidase development in the cold. Addition of gibberellic acid stimulated indoleacetic acid oxidase development during germination at room temperature and during cold treatment. Amo-1618 inhibited indoleacetic acid oxidase development before and during cold treatment. Indoleacetic acid treatment increased indoleacetic acid oxidase activity during germination at room temperature while no significant effect on activity was observed during cold treatment.     

小麦生育前期POD同工酶的动态变化

利用复性电泳技术 ,对 2个小麦品种 (冬性品种、春性品种 )从出苗到拔节初期绿叶中的POD酶谱进行分析 ,结果表明 :( 1)小麦从出苗到拔节初期 POD的分子量均在 35k D以上 (含 35k D) ;( 2 )三叶期以前叶片中 POD活性比三叶期以后的弱 ;( 3)小麦叶片中 POD酶带变化主要集中在 52～ 146k D间 ;( 4 )从出苗到拔节初期小麦绿叶中始终含有 30 0、 52、 39、 37k D4条活性较强的过氧化物酶 ;( 5)在不同的生育阶段和环境条件下 ,绿叶中 POD的种类和活性均有显著变化。     

Effect of d-mannose on antioxidant defense and oxidative processes in etiolated wheat coleoptiles

Effect of d-mannose treatment on different antioxidants, phenolics, protease activity, lipid peroxidation, DNA damage and cell death was investigated in coleoptiles of etiolated wheat seedlings. Modulations in these biochemical parameters were monitored up to 96 h after treatment at 24 h intervals. With accelerating effect on initial signs of cell death, i.e., appearance of long DNA fragmentation and no effect on initiation of terminal stage, i.e., internucleosomal nDNA fragmentation, mannose treatment (1 % = 56 mM) diminished the antioxidant activities in wheat coleoptiles. Mannose treatment decreased the catalase activity at all intervals, while APX and POD activities decreased at 72 h. Peroxidation of lipids increased at 72 h after mannose treatment. Levels of most of antioxidants, i.e., SOD, peroxidases and phenolics were raised during initial time period (24–48 h) of mannose treatment probably as an attempt to counter the stress effect. Protease activity gradually increased and protein content decreased with time in both treated and non-treated coleoptiles. Sharp decrease in CAT, APX and peroxidase activities and increase in lipid peroxidation at 72 h overlaps with apoptotic internucleosomal nDNA fragmentation in this organ. This coincidence points towards the importance of compromised antioxidant defense and involvement of reactive oxygen species in initiation of terminal stage of programmed cell death in wheat coleoptile. In conclusion, accelerating effect on DNA fragmentation and lipid peroxidation along with diminished antioxidant activities at the time of internucleosomal nDNA fragmentation, provide evidence for pro-apoptotic effect of d-mannose in wheat coleoptile.     

Effect of manganese toxicity on the indoleacetic Acid oxidase system of cotton

The effect of substrate manganese on tissue manganese levels and activity of the indoleacetic acid (IAA)-oxidase system of cotton (Gossypium hirsutum, L.) was investigated. A sand culture technique was used with 1, 3, 9, 27 and 81 mg manganese (MnSO₄) per liter nutrient solution applied in various experiments.

The following relationships held for both long-term (126 days) and short-term (12-14 days) exposures to manganese treatment: A) There was a direct relationship between substrate and tissue manganese. B) Only the 81 mg/liter Mn plants exhibited severe manganese toxicity symptoms. C) At the toxic level of manganese an increased IAA-oxidase activity and decreased IAA-oxidase inhibitor activity was observed. There was a direct relationship between degree of enzyme response and severity of visible symptoms. D) With the manganese toxicity plants, but none of the other treatments, extracts of the young leaves contained as much IAA-oxidase activity as extracts of much older leaves. E) Crude extracts from the plants grown with 81 mg manganese per liter solution, in contrast to those of other treatments, destroyed IAA without addition of MnCl₂ to the assay medium.

A hypothesis is advanced stating that manganese toxicity symptoms in cotton are expressions of auxin deficiency caused by IAA-oxidase activity increased by the abnormal tissue levels of manganese.