Anions activate the oxidation of indoleacetic Acid by peroxidases from tomato and other sources

Anionic peroxidase from tomato (Lycopersicon esculentum) fruit oxidized indoleacetic acid (IAA) slowly in the presence of Mn²⁺ and dichlorophenol in acetate buffers. The addition of certain anions to the reaction mixture increased the rate of oxidation. Phosphate was one of the effective anions and exerted maximal activation at 0.1 molar. The most effective activator of tomato peroxidase was nitrilotriacetate (NTA) at an optimum concentration of 60 micromolar. Only 0.17 nanomolar peroxidase was needed to oxidize 0.1 micromole IAA/5 minutes in the presence of NTA compared to 650 nanomolar peroxidase for the same rate in the absence of NTA. Other effective anions were oxalate, pyrophosphate, malate, and citrate. Each activator exhibited an optimum concentration and higher concentrations were inhibitory. Anionic peroxidase from horseradish was activated by the same anions. A cationic peroxidase from horseradish and lactoperoxidase oxidized IAA in acetate buffer although anions activated these enzymes severalfold. Microperoxidase and other hematoporphrins also catalyzed IAA oxidation in the presence of anions. It is proposed that IAA oxidation by peroxidase may be important when vacuolar contents mix with peroxidase as during plant injury.     

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn²⁺ and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H₂O₂, were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mμ was reduced, while major absorption peaks appeared at 254 and 247 mμ. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mμ. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H₂O₂ (3 μmoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H₂O₂ inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H₂O₂ (0.03 μmole/ml). A comparison of K_m values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.     

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.     

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.     

Saturable uptake of indol-3yl-acetic Acid by maize roots

The uptake of 5-[³H]indol-3yl-acetic acid (IAA^*) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K_m of 0.285 micromolar and apparent V_max 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA^* took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.     

Rapid effects of indoleacetic Acid and ethylene on the growth of intact pea roots

Root auxanometers were used to determine the growth rates of individual intact primary roots accurately and quickly. The growth of pea (Pisum sativum L.) roots was inhibited by both indoleacetic acid and ethylene within 20 minutes. A supramaximal concentration of ethylene inhibited root growth less than did 5 to 20 mum indoleacetic acid, indicating that inhibition of root growth by auxin was not due only to indoleacetic acid-induced ethylene production. Inhibition of root growth was largely relieved within 60 minutes of removal of both growth regulators.     

Production of peroxidases by hairy roots ofBrassica napus

Hairy roots cultures derived from leaf explants ofBrassica napus L. produced and secreted peroxidases. The enzyme activity in the medium increased with growth but it remained nearly constant in the tissue. The changes in extracellular peroxidase activity seemed to be correlated with the increase in a basic peroxidase of pI: 9.6. Four isoenzymes with pI in the range 8.5–9.6 and a neutral peroxidase of pI 6.3 were the most important peroxidases detected in cell extracts. Ca²⁺ addition at the beginning of the culture stimulated both the excretion of peroxidase to the medium and the enzyme activity in hairy roots but the isoenzyme profiles did not show qualitative changes during the growth cycle for both culture conditions.     

Properties of anionic peroxidases purified from Chinese cabbage roots

Two anionic peroxidases were isolated from Chinese cabbage roots and purified using gel filtration followed by ion-exchange chromatography. Following purification a specific activity of peroxidases was estimated as 50 units.mg^-1 (A1) and 30 units.mg^-1 (A2) compared with that of a crude extract of the peroxidases which was 2.31 units.mg^-1. The pH for its optimum activity was 5.0 and the addition of Ca²⁺ produced a 15 % increase in peroxidase activity. Isoelectric focusing techniques were carried out in order to classify the peroxidases based on their isoelectric point (pI). Two anionic peroxidases, A1 and A2, were found to have pI values of 4.83 and 4.78, respectively. The peroxidases were found to be heat-stable, with 20 % (A1) and 16 % (A2) of the enzymatic activity remaining after heat treatment at 70 °C for 20 min. The heat inactivation rate followed first-order kinetics with the activation energy; Ea, estimated as 38.2 kcal.mol^-1 and 36.4 kcal.mol^-1 for A1 and A2, respectively.     

One- and two-electron oxidation of reduced glutathione by peroxidases

The oxidation of glutathione by horseradish peroxidase forms a thiyl free radical as demonstrated with the spin trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the radical trap 5,5-dimethyl-1-pyrroline-N-oxide. In contrast to L-cysteine oxidation, glutathione oxidation is highly hydrogen peroxide-dependent. The oxidation of glutathione by glutathione peroxidase forms glutathione disulfide without forming a thiyl radical intermediate, except in the presence of the thiyl radical-generating horseradish peroxidase.     

In vitro uptake and processing of prezein and other maize preproteins by maize membranes

A cell-free, mRNA-dependent system has been developed for the translation and processing of zein preproteins. A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages. When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place. The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently. The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent. This small uptake, or absorption, or unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein. Finally a sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system has been developed which is particularly suited for the separation of low molecular weight proteins (less than 10,000 daltons). Using this method, we examined the products of in vitro zein processing and detected no presequence polypeptides. This suggests that the zein cleavage proteinase is probably an exopeptidase.     

Purification and measurement of abscisic Acid and indoleacetic Acid by high performance liquid chromatography

A procedure was selected for the simultaneous extraction and purification of abscisic acid (ABA) and indoleacetic acid (IAA). Unnecessary steps were eliminated and an accumulation of aqueous phase was avoided. The superior performance of diethyl ether (compared to ethyl acetate) for bulk purification and the superior resolution provided by 250 millimeter columns packed with 5-micrometer spherical particles of strong anion exchanger and octadecylsilane (C18) greatly facilitated the purification of samples. A fixed-wavelength (254 nanometer) ultraviolet detector and a fluorescence detector connected in series on a high performance liquid chromatograph permitted nondestructive monitoring and measurement of ABA and IAA. Derivatization was not necessary for chromatography or for detection. Isocratic elution with simple mobile phases gave sharp peaks. A few simple precautions minimized losses. Recoveries through the entire procedure averaged about 75% for ABA and about 50% for IAA. Purified ABA and IAA fractions were usually free of interfering contaminants. Identities were confirmed by gas chromatography-mass spectrometry.     

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.     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

In vitro synthesis of zein-like protein by maize polyribosomes.

Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes $\text{in vitro}$ produced one main radioactive protein. This protein was soluble in 70% ethanol and had the same mobility in electrophoresis on sodium dodecyl sulfate-gels as a zein standard. The ratio of [¹⁴C] leucine to [¹⁴C] lysine incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membrane-bound polyribosomes.     

Immunological characterization of soluble peroxidases from rat tissues including preputial gland

A highly active soluble peroxidase has been identified in the preputial gland of rats and characterized immunologically along with other soluble peroxidases of a number of rat tissues such as submaxillary gland, exorbital lacrimal gland and also of the uterine fluid of the estrogen treated rats. All these peroxidases have the native molecular weight around 73K as determined by gel filtration on Sephadex G-150. An antiserum raised against the pure bovine lactoperoxidase interacts with all these soluble peroxidases and immunoprecipitates the enzyme activity in a similar fashion when titrated against varied concentration of the antiserum. Following electrophoretic transfer to nitrocellulose by Western blotting, the antiserum crossreacts with the preputial, submaxillary and lacrimal gland protein of molecular weight around 73K and with the uterine fluid protein of molecular weight of 80K. An additional crossreacting protein of molecular weight of 80K is also evident in the lacrimal gland. All these enzyme preparations, however, contain another immunoreactive protein of molecular weight of about 64K. While 73–80K molecular weight interacting proteins may represent different forms of peroxidase, presumably with varied carbohydrate moieties, 64K molecular weight protein may be a precursor of the peroxidase which after posttranslational modification such as heme conjugation and glycosylation leads to formation of native enzyme. Rat harderian gland, unlike bovine origin, does not contain any detectable peroxidase activity. The immunoblot does not show the presence of any immunoreactive protein around 73K except the 64K molecular weight protein indicating that this gland can not synthesize the native peroxidase from this precursor probably due to some block in posttranslational modification.     

One-electron oxidation of Trolox C and vitamin E by peroxidases.

Direct reactions of peroxidases with Trolox C (a vitamin E analogue) and vitamin E were observed in 50% (v/v) methanol. The kinetic results revealed that the reaction of horseradish peroxidase intermediate Compound II with Trolox C and vitamin E was the rate-determining step, and the rate constants were estimated to be 1.7 x 10(3) and 5.1 x 10(2) M-1.s-1, respectively. Peroxidases catalyzed the one-electron oxidation of Trolox C and vitamin E, and the vitamin E phenoxyl radicals resulting from the peroxidase reactions were detected by continuous-flow ESR spectroscopy.     

Changes in soluble amino-N,soluble proteins and free amino acids in leaves and roots of salt-stressed maize genotypes

Abstract

The effects of salt stress on the contents of organic solutes and on the pattern of free amino acids were studied in leaves and roots of two maize genotypes, BR5033 (salt-tolerant) and BR5011 (salt-sensitive). In leaves and roots of salt-stressed plants, soluble amino-N increased with time when compared to the controls. Salt stress increased the soluble protein content only in leaves of BR5011. Salinity increased the content of the majority of the free amino acids in leaves and roots of genotypes studied. Results suggest the hypothesis of disturbances in translocation of N-containing compounds from shoot to root in the salt-sensitive genotype. Results also suggest that the accumulation of organic solutes, mainly in roots of BR5033, may have an important role in the tolerance of this genotype to salt stress.     

Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro

Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The “survival” of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues.     

Membrane-bound class III peroxidases: identification, biochemical properties and sequence analysis of isoenzymes purified from maize (Zea mays L.) roots

The occurrence of three plasma membrane-bound class III peroxidases has been demonstrated for maize (Zea mays L.) roots [Mika and Lüthje (2003) Plant Physiol. 132:1489-1498]. In the present work a novel PM-bound peroxidase (pmPOX3) was partially purified. The experimental molecular mass of the heme protein was 38 kDa after size exclusion, and 57 kDa in non-reducing SDS-PAGE stained with the peroxidase substrates tetramethylbenzidine and H(2)O(2). The glycosylation of pmPOX1, pmPOX2b and pmPOX3 was shown by different approaches. The full length sequences of pmPOX1, pmPOX2b and pmPOX3 were identified by ESI-MS/MS and MALDI-TOF MS analysis in combination with in silico and in vivo cloning. Thus, we report the first sequence analysis of membrane-bound class III peroxidases. A partial gene analysis revealed two or three introns. Experimental and theoretical isoelectric points and molecular masses were compared. Targeting signals, the putative protein structures and the localization of the active center of the enzymes on the outside of the plasma membrane were deduced of the amino acid sequences. In contrast to other class III peroxidases, pmPOX1 seems to have a dimeric structure. Predictions of hydrophobic domains in comparison with solubilization experiments suggest an N-terminal transmembrane domain for the isoenzymes.     

Differentiation between indoleacetic Acid and the citrus auxin by column chromatography

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.