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.     

Localization and General Properties of Developing Peach Seed Coat and Endosperm Peroxidase Isoenzymes

Changes of soluble and ionically bound peroxidase and indoleacetic acid (IAA) oxidase activities were followed during peach seed development. Soluble peroxidase activity was located mainly in the embryo plus endosperm tissue, whereas wall ionically bound activities were found predominantly in the integument tissue. The different peroxidase isoenzymes present in the extracts were characterized by polyacrylamide gel electrophoresis and isoelectric focusing; the main soluble isoenzyme of embryo plus endosperm tissue was an anionic isoperoxidase of R _F 0.07. Basic ionically bound isoenzymes were located only in the integument tissue, but two soluble anionic isoenzymes of R _F 0.23 and 0.51 were also present in this tissue. In parallel, peroxidase protein content was estimated specifically using polyclonal antibodies. The kinetic data and the changes of seed IAA oxidase activity during fruit development suggested that basic peroxidase isoenzymes from ionically bound extracts of integument might be involved in IAA degradation. Received September 11, 1997; accepted October 21, 1997     

Cytokinin-controlled Indoleacetic Acid Oxidase Isoenzymes in Tobacco Callus Cultures

Indoleacetic acid oxidase in tobacco callus tissues (Nicotiana tabacum L., cultivar White Gold) was resolved into seven anionic isoenzymes by polyacrylamide gel disc electrophoresis. Different concentrations of kinetin and zeatin in the presence of indoleacetic acid affected the level of this enzyme, particularly two fast-moving isoenzymes, A₅ and A₆. The optimal concentration of kinetin was 0.2 μm; increasing concentrations above this level progressively lowered the total activity of indoleacetic acid oxidase and repressed the development of isoenzymes A₅ and A₆. Actinomycin D and cycloheximide inhibited the development of these two isoenzymes under the influence of 0.2 μm kinetin, suggesting a requirement for RNA and protein synthesis. The cytokinin-promoted indoleacetic acid oxidase isoenzymes A₅ and A₆ increased with time and paralleled the dry weight increase of tobacco callus tissues, but the total activity of indoleacetic acid oxidase per unit dry weight of tobacco callus varied with time depending on the stage of plant growth.     

On Extraction and Quantitation of Plant Peroxidase Isoenzymes

Peroxidase in tobacco callus tissue differed in extract-ability depending on the subcellular distribution of the enzyme. Based on extractability it consisted of four fractions: freely soluble and less freely soluble in phosphate buffer, KCl-soluble, and insoluble. The latter two fractions were un-extractable by a phosphate buffer alone. The different fractions contained varied proportions of peroxidase isoenzymes. The extractability of indoleacetic acid oxidase was similar. A medium of high ionic strength is essential for quantitative extraction of peroxidase and indoleacetic acid oxidase isoenzymes. For quantitation of isoperoxidase activity on polyacryl-amide gel following electrophoretic separation, benzidine and o-dianisidine were better hydrogen donors than guaiacol and pyrogallol. The optimum pH was 4.5, but a citrate buffer was inhibitory. The optimum conditions included an acetate buffer at pH 4.5, a substrate concentration of 0.03 %, benzidine as the hydrogen donor, and a 3-minute treatment with 7 % acetic acid after staining. The color intensity of the bands remained unchanged for at least three days. With appropriate sample size and reaction time there was a linear relationship between enzyme concentration and activity.     

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.     

Activation of Avena coleoptile cell wall glycosidases by hydrogen ions and auxin

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of β-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo β-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.     

Lignin formation in wheat coleoptile cell walls: a possible limitation of cell growth

Four growth-influencing compounds—hydroxyproline, 2,2′-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid—were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth.     

The relationship of the peroxidative indoleacetic Acid oxidase system to in vivo ethylene synthesis in cotton

Since peroxidase and manganese have been implicated in both auxin destruction and ethylene production, the effect of auxins and high tissue levels of manganese on the peroxidative indoleacetic acid oxidase system and the internal level of ethylene was determined in cotton (Gossypium hirsutum L. cv. Watson GL-7). The highest level of manganese tested produced manganese toxicity symptoms, including necrotic lesions, accompanied by an increase in internal ethylene levels at about 15 days after treatment initiation. Statistically significant increases in indoleacetic acid oxidase and peroxidase activity were first observed 2 days later and were paralleled by tissue manganese levels above 7.4 milligrams per gram dry weight and internal ethylene levels of 0.77 microliters per liter air. Eight hours after application of 2,4-dichlorophenoxyacetic acid or indoleacetic acid, the internal levels of ethylene were increased to above 6.6 microliters per liter air in cotton plants, and levels of this magnitude were maintained for a 72-hour period of observation. Modification of peroxidase and indoleacetic acid oxidase activity in auxintreated plants definitely occurred well after the elevation of internal ethylene levels. While ethylene levels and indoleacetic acid oxidase activity were increased by both experimental approaches, the earlier appearance of increased ethylene indicates that the peroxidative indoleacetic acid oxidase system in cotton is not involved in ethylene synthesis or that this enzyme is not the rate-limiting factor when ethylene synthesis is increased. Ethylene, as well as auxin destruction, may be involved in some of the long term plant responses to toxic levels of manganese. The findings also suggest that auxin-induced ethylene may play a role in the elevation of peroxidase and indoleacetic acid oxidase activity eventually seen in extracts of plants treated with auxins. The data support the assumption that the enzymatic portion of the indoleacetic acid oxidase system in cotton is a peroxidase.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

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.     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Metabolism of Indole-3-Acetic Acid: II. Oxindole Pathway in Parthenocissus tricuspidata Crown-Gall Tissue Cultures

An indoleacetic acid oxidase preparation from an acetone powder of Parthenocissus tricuspidata crown-gall tissue has been examined. An intermediate in the reaction is 3-hydroxymethyloxindole and nonenzymic conversion of it to 3-methyleneoxindole was observed. Neither reaction mixtures nor 3-methyleneoxindole have any auxin-like activity in Avena or wheat coleoptile bioassays. In vivo studies show that although 53% decarboxylation of indoleacetic acid was observed in 48 hours, only a small amount of 3-methyloxindole could be recovered from the medium. The other decarboxylated products remain to be identified but are not 3-hydroxymethyloxindole or 3-methyleneoxindole.     

Studies on indoleacetic acid oxidation by liquid medium from crown gall tissue cells: The role of malic acid and related compounds

1.

1. Indoleacetic acid oxidation by liquid medium from crown gall tissue culture cells has been studied. The reaction has a pH optimum of 4.5 and requires Mn²⁺ and a monohydric phenol. A short lag phase is routinely observed. The appearance of peroxidase and indoleacetic acid oxidising activity in the medium of a tissue culture was followed over a 3 week time course. One function of this enzyme may be to prevent the accumulation of excess inhibitory concentrations of indoleacetic acid.     

Electrophoretic study on peroxidase,indoleacetic acid oxidase,and o-diphenol oxidase fractions in extracts from different growth zones ofvicia faba L. roots

Using electrophoresis in acrylamide gel, fractions of peroxidase, indoleacetic acid oxidase, and o-diphenol oxidase were investigated in extracts from three growth zones ofVicia faba L. roots. Three peroxidase fractions (zones) moving towards the anode were revealed as well as four peroxidase fractions (zones) migrating towards the cathode. Three peroxidase fractions showed detectable indoleacetic acid oxidase activity. The o-diphenol oxidase activity was revealed in all peroxidase fractions moving towards the anode, in those moving towards the cathode the o-diphenol oxidase activity differred according to the substrate used. One fraction with both peroxidase and o-diphenol oxidase activity occurred only in electrophoreograms of extracts from the maturation zone; in this fraction no indoleacetic acid oxidase activity was demonstrable.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

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     

PEROXIDASE LOCALIZATION,ACTIVITY, AND ISOZYME PATTERNS IN THE DEVELOPING SEEDLING OF VANDA (ORCHIDACEAE)

Peroxidase activity in the seedling of Vanda was investigated at various stages of development. Active sites were demonstrated histochemically and soluble proteins and peroxidase isozymes were resolved by disc electrophoresis at progressive stages of growth. Activity, which is highest in the early stages of development and lowest at the stage when maximum differentiation occurs, is confined to the epidermal layer and outer surface of the seedling in early stages of development, It is also present in the vascular tissues of the leaves, root, and parenchymatous region at later stages. Indoleacetic acid raises peroxidase activity when present in the growth medium in physiological concentrations. The number of isoperoxidases varies with developmental stage and is lowest in the stage at which leaves and roots are initiated. These observations are discussed in relation to the hypothesis that the peroxidases play a role in morphogenesis as a part of the indoleacetic acid-oxidase system.     

Auxin transport in Avena: I. Indoleacetic Acid-C distributions and speeds

Measurements have been made on the initial stages of the transport of carbon-14-labeled indoleacetic acid in the coleoptile of Avena sativa L. Concentrations of mobile and immobilized indoleacetic acid are related to both distance and time during the first 2 hours after application of the indoleacetic acid at several concentrations to the top of the decapitated coleoptile.     

Comparative aspects of root and root nodule secondary metabolism in alfalfa

l-phenylalanine ammonia lyase (PAL), indoleacetic acid oxidase (IAA oxidase), O-methyltransferase, peroxidase, total phenolics and total indoles were compared in roots and root nodules of alfalfa. PAL, O-methyl-transferase, total phenolics and total indoles were higher in nodules than in roots. Isolated bacteroids were assayed for O-methyltransferase, PAL, peroxidase and total phenolics, but their levels were either low or not detectable. Nodule leghemoglobin was separated by disc gel electrophoresis and found to have IAA oxidase activity. Phenolics, IAA oxidase and leghemoglobin appear to be interrelated in regulating indole levels in the nodule.