Changes in Indoleacetic Acid Oxidase Activity Associated with Plant Water Potential

The effect of plant water potential on the activity of indoleacetic acid oxidase was examined. It was found that with increasing plant water deficit the activity of indoleacetic acid oxidase increased. Higher activies of this enzyme are known to be associated with older tissues and lower endogenous auxin levels. It is suggested that while water stress may adversely affect a variety of physiological processes, increases in the activity of indoleacetic acid oxidase may provide plants with a drought adaption mechanism.     

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.     

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.     

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.     

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.     

Effect of indoleacetic Acid and hydroxyproline on isoenzymes of peroxidase in wheat coleoptiles

Indoleacetic acid at 0.017 millimolar inhibited the formation of three peroxidase isoenzymes in both soluble and wall-bound enzyme fractions of wheat coleoptile (Triticum vulgare) tissue. Hydroxyproline at 1 millimolar prevented the indoleacetic acid-induced inhibition. Indoleacetic acid oxidase activity in the soluble fraction was decreased by indoleacetic acid and was restored by hydroxyproline. Most of the indoleacetic acid oxidase activity was located in the electrophoretic zones occupied by two of the peroxidase isoenzymes influenced by indoleacetic acid and hydroxyproline. At least part of the effect of hydroxyproline on auxin-induced elongation of coleoptile tissue may be through control of auxin levels by indoleacetic acid oxidase.     

光叶楮扦插生根的吲哚乙酸氧化酶、多酚氧化酶、过氧化物酶活性变化研究

对光叶楮扦插生根过程中吲哚乙酸氧化酶（IAAO）、多酚氧化酶（PPO）、过氧化物酶（POD）3种酶进行了动态跟踪分析。结果表明：IAAO活性在扦插初期逐渐上升,第10d上升到高峰,之后下降再上升,第30d达到新高峰,然后迅速下降;前25d POD活性变化规律与IAAO相似,但30d以后活性一直上升;PPO活性在扦插前期缓慢上升,第20d上升到了最高点,此后变化不大。还研究了IAAO、PPO、POD与不定根的发生和发展关系,认为光叶楮扦插生根可分为愈伤组织形成期、根诱导期和根的伸长期3个阶段,愈伤组织形成期3种酶活性都呈上升趋势,根诱导期IAAO和POD的活性达到高峰;而根伸长期IAAO和POD活性下降,PPO活性上升。     

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.     

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.     

Oxidative stress and formation and maintenance of root stem cells

The hypothesis of L. Feldman and his coworkers, according to which a more oxidizing environment in the cells of root quiescent center results from high activity of ascorbate oxidase activated by indoleacetic acid (IAA) accumulating in these cells, is discussed. The high activity of ascorbate oxidase is responsible for lowered concentrations of the reduced form of ascorbic acid and glutathione and high content of reactive oxygen species in quiescent center cells. The oxidative stress represses proliferation of the cells. Inhibitors of IAA transport attenuate the oxidative stress, thus suggesting a role of IAA as an activator of ascorbate oxidase. Interestingly, the high concentration of IAA in dividing cap cells adjacent to the quiescent center cells did not cause retardation of cell proliferation and oxidative state in these cells.     

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.     

The Promotion of Indole-3-acetic Acid Oxidation in Pea Buds by Gibberellic Acid and Treatment

Terminal buds of dark-grown pea (Pisum sativum) seedlings have an indole-3-acetic acid oxidase which does not require Mn(2+) and 2,4-dichlorophenol as cofactors. Oxidase activity is at least 50 times higher in buds of tall peas than in dwarf seedlings. Administration of gibberellic acid to dwarf peas stimulates both growth and indoleacetic acid oxidase activity to the same levels as in tall seedlings. By contrast, indoleacetic acid oxidation assayed in the presence of Mn(2+) and 2,4-dichlorophenol proceeds at similar rates regardless of gibberellin application. Treatment of tall peas with the growth retardant AMO-1618 reduces growth and oxidase activity. Such treated seedlings are indistinguishably dwarf. The enzyme does not appear to be polyphenol oxidase, nor do the results suggest that reduced activity in dwarf buds is due to higher levels of a dialyzable inhibitor. The peroxidative nature of the oxidase is probable.     

Effects of Pargyline on Mitochondrial Amine Oxidase Activity, Indoleacetic Acid Synthesis and Growth of Crown-Gall Tumor Callus

Vinca rosea L. crown-gall tumor callus tissue cultures treated with N-benzyl-N methyl propargylamine (pargyline) exhibited a decrease in the level of endogenous indoleacetic acid from 0.42 μg/mg of protein to less than 0.30 μg/mg of protein. A simultaneous decrease in the specific activity of mitochondrial amine oxidase from 3000 units to less than 250 units at 1.0 μM, 0.01 mM, 0.1 mM and 1.0 mM pargyline, suggested a relationship between amine oxidase function and indoleacetic acid synthesis. Tryptamine incorporation into indoleacetic acid was also decreased at these concentrations. Pargyline inhibited tumor callus growth significantly (based on fresh weight measurements) at the highest concentration, 1.0mM. These data support the hypothesis of a coordinate metabolic system linking mitochondrial amine oxidase activity and indole acetic acid synthesis. Inhibitory action of pargyline on the enzyme is reflected in reduced indoleacetic acid levels and, ultimately, in reduced callus growth rates.     

Chromatographic degradation of phloridzin

Phloridzin, the main phenolic glucoside in apple leaves, has been found to undergo transformation during chromatography. When chromatographed repeatedly in ammoniacal solvents, at least 2 new derivatives appeared. One of these was identified as phloretic acid. When bioassayed in the presence of indole-3-acetic acid this substance behaved as though it promoted the destruction of the auxin. Comparative bioassay with naphthaleneacetic acid suggested that phloretic acid acts on indoleacetic acid destruction via stimulation of indoleacetic acid oxidase. However, at low concentration and in presence of a small amount of phloridzin it also showed a synergistic effect with indoleacetic acid.

A substance with the same characteristics was obtained directly from apple leaves, which are known to contain phloridzin when the extracts were chromatographed only once in the same (alkaline) solvent. While not completely confirmed, this suggests that phloretic acid is normally present in apple leaves, where it may affect growth there by promoting indoleacetic acid oxidation.

     

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.     

噻二唑苯基脲在平贝母脱分化过程中引起的生理生化变化

平贝母(Fritillaria ussuriensis Maxim)鳞茎切块分别在含噻二唑苯基脲(TDZ)或激动素(Kin)的培养基上脱分化过程中的苯丙氨酸解氨酶、淀粉酶、吲哚乙酸氧化酶和过氧化物酶活性均增强,在外植体脱分化前达到峰值,随后下降。TDZ处理下这些酶的活性均高于Kin处理。可溶性蛋白含量的变化趋势与酶活性变化相同,只是TDZ作用下蛋白含量增加的相对水平较低。说明TDZ具有比Kin更强的细胞分裂素活性。     

Stimulation of indoleacetic Acid oxidase and inhibition of catalase in cotton extracts by plant acids

Activity of indoleacetic acid oxidase in partially purified extracts from cotton is stimulated by small amounts of malate, succinate, fumarate, and other plant acids. The stimulation is apparently due to inhibition of catalase, which is detectable in certain preparations. The lag phase of indoleacetic acid oxidation by crude preparations is eliminated by steps in processing which conceivably either denatures or dilutes catalase, or concentrates inhibitors to catalase.     

HORMONAL REGULATION OF EPIPHYLLOUS BUD RELEASE AND DEVELOPMENT IN BRYOPHYLLUM CALYCINUM

The effects of various concentrations of several auxins and cytokinins separately and in combination on epiphyllous budding of Bryophyllum calycinum were investigated, using a marginal leaf strip method. The number of buds released per leaf in 10⁻⁶ m benzyladenine (BA) was 2.8 times greater than those in water, and 1.6 times greater in 10⁻⁵ m isopentyladenine (IPA). BA, IPA and kinetin were antagonistic to indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) when used in combination treatments. The IAA oxidase co-factor p-coumaric acid (PCA) increased the number of epiphyllous buds to twice that of the water controls. This stimulatory effect of PCA on bud release suggests that an increase in IAA oxidase activity may be one of the significant changes that precedes the release of epiphyllous buds.     

On the Physiological Effects of Phloridzin, Phloretin and some Related Substances upon Higher Plants

Some dihydrochalcones and related flavonoids were tested Cor their effects upon indoleacetic acid oxidase (mainly from pea roots) and upon oxidative phosphorylation in mitochondria from cucumber hypocotyls. Also their influence upon the growth of wheat roots and upon the absorption of ¹⁴C-labelled sugars and 2, 4-dichloro-phenoxyacetic acid in wlieat roots was studied. The dihydrochalcone glucoside phloridzin known for its strong inhibitory effects upon sugar transport in some animal tissues gives no corresponding specific effects in higher plants. Phloridzin and some related dihvdrochalcone glycosides as well as the structurally similar compounds naringenin and 2′, 4, 4′-trihydroxychalcone are potent stimulators of indoleacetic acid oxidase. Sieboldin, which compared to phloridzin has an additional hydroxyl group (giving an ortho-diphenolic substitution in the B-ring) is a strong inhibitor of indoleacetic acid oxidase (10^?6M gives about 50 per cent Inhibition). Phloridzin and sieboldin replace each other in different groups of species in the genus Malus. The aglycones phloretin, naringenin and 2′, 4, 4′-trihydroxychalcone stimulate wheat root growth (especially in the presence of auxins or growth-inhibilory sugars) and inhibit the absorption of sugars and of 2, 4-dichlorophenoxyacetic acid. They also inhibit oxidative phosphorylalion and give a distinct uncoupling effect. The glycosides are as a rule less active in all these respects and sieboldin is almost inactive. There is a rather good correlation between uncoupling of oxidative phosphorylation and effects upon root growth, whereas there is no agreement in the details between the effects upon root growth and upon IAA oxidase.     

Ontogeny and Hormonal Control of Polyphenoloxidase Isozymes in Tobacco Pith

Isozymes of tobacco pith polyphenoloxidases (o-diphenol oxidase, EC 1.10.3.1) were separated electrophoretically from fresh pith of intact plants and from cultured pith sections. Extracts of fresh pith contained a poorly resolved complex of two to three anodic bands after starch gel electrophoresis at alkaline pH. This anodic complex was more active with chlorogenic acid than with 3,4-dihydroxyphenylalanine and was found in greater activity per gram fresh weight of tissue in younger internodes than in older ones. The longitudinal gradient of activity was thus the opposite of that found for the constitutive isozymes of peroxidase.A well defined cathodic band of polyphenoloxidase activity appeared after culture of pith in modified White's medium with shaking. This band, which was more active with 3,4-dihydroxyphenylalanine than with chlorogenic acid, could be detected after 1 to 2 days of incubation. Its appearance was enhanced by the addition of 10 mum indoleacetic acid; kinetin (1 mum tended to prevent this indoleacetic acid effect). Such hormonal control is opposite to that previously reported for the rapidly appearing new isozymes of peroxidase.The pattern of the major isozymes associated with polyphenoloxidase activities differs from that of peroxidase.