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.     

The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala.

1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.     

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.     

Electrophoretic investigation of ribonuclease in roots and leaves ofVicia faba L.

Isozyme patterns and specific activity of ribonuclease (ribonucleate pyridinenucleotido-2′-transferase, E. C. 2.7.7.16) were followed in the extracts of segments from three growth zones of the root and in extracts of young and senescent leaves ofVicia faba L. Electrophoreograms of extracts from all three investigated root zones were identical, in the electrophoreograms of extracts from senescent leaves however one new ribonuclease occurred which could not be detected in the electrophoreograms of extracts from young leaves. Extracts from senescent leaves had higher specific activity of ribonuclease than extracts from young leaves. Extracts from the enlargement zone of the root and those from the maturation zone had a three times higher specific activity of RNase than extracts from the division zone.     

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.     

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.     

The effect of water stress on indoleacetic Acid oxidase in pea plants

Activity of indoleacetic acid oxidase was shown to increase following a period of water stress. Two fractions of indoleacetic acid oxidase were extracted from plant extracts. Similarly, two protein peaks (determined by ultraviolet absorption) were isolated. One peak, associated with an indoleacetic acid oxidase peak, increased following water stress. The second peak, not associated with extractable indoleacetic acid oxidase, decreased after water stress. The results are discussed in terms of general growth effects.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

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.     

Glucose-6-phosphate dehydrogenase isoenzymes in root growth zones ofVicia faba

The specific activity of glucose-6-phosphate dehydrogenase (G-6-PD) in growth zones ofVicia faba roots is increasing with cell maturation and differentiation. Changes in the total activity of G-6-PD are not associated with a change in the number of G-6-PD isoenzymes. Five G-6-PD isoenzymes were found in all root growth zones. Some differences were found in the activity of individual isoenzymes.     

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.     

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.     

Irradiations à faible énergie et biosynthèse d'amarantine chez des plantules d'Amarantus tricolor L. var. bicolor ruber Hort.

Summary It has been shown that indoleacetic acid (IAA) does not occur in developing grains of Hordeum vulgare L. (barley), but that an unidentified indolic compound does. This compound, designated A, was also found to be a product of the metabolism of exogenous IAA by barley. The expression of the gibberellic acid effect was delayed for at least 8 h if grains were imbibed in a solution of IAA, and during this time, the IAA was metabolised. The enzyme system involved could be peroxidase, which was active in the grains at all stages of their development and at maturity, and partially purified extracts of peroxidase were found to have considerable IAA oxidase activity.Abbreviations DMABA dimethyl aminobenzaldehyde - dMACA Dimethyl amino cinnamylaldehyde - GA gibberellic acid - IAA indole acetic acid     

Peroxidase,polyphenol oxidase and catalase isoenzymes during germination and early plant development of tall and dwarf wheats (Triticum aestivum L.)

Isoenzyme pattern of peroxidase, polyphenol oxidase and catalase at different intervals during germination and in different plant parts during early development of ‘C-306’ and ‘Hira’ cultivars, representing tall and dwarf wheats, respectively were investigated. The number of isoenzymic components of the three enzymes increased progressively with the concomitant increase in the stage of germination. The isoenzymic patterns, in general, were more clear and better developed during the later stages of germination. Different tissues or organs of the wheat plant possessed specific isoenzymic pattern of these enzymes. Differences between cultivars were observed both in the number and relative intensity of the various isoenzymic fractions.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Isoenzymes of lactate dehydrogenase in the root growth zones ofVicia faba L.

The present studies of the LDH isoenzymes have been made as a part of our studies on the respiration systems of the roots ofVicia faba. All the root zones had 5 LDH isoenzymes and 2 antibands, but there were differences in their relative quantity. Cathodic isoenzymes (LDH₅, LDH₄) prevailed in the meristematic zone, whereas the anodic ones (LDH₁) prevailed in the others. The different enzyme activity in individual zones is probably connected with the transition of the anaerobic metabolism in the meristem to the aerobic one in the differentiating parts of the root.     

Contribution to the estimation of malic dehydrogenase isoenzymes in the root growth zones ofVicia faba L.

Highly active NAD-MDH (E.C.1.1.1.37) and low activity of NADP-MDH (E.C.1.1.1.40) were found inVicia faba roots. The NAD-MDH activity is associated with 6 to 12 isoenzymes. The number of isoenzymes is dependent on the extraction (use of Triton X-100etc.) and detection procudures (presence of KCN, phenazine methosulphate). The meristematic zone does not contain one isoenzyme (X) which is present in the other two zones. The meristematic zone, elongation zone and zone with the beginning differentiation differ in their activity of individual isoenzymes.     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.