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.     

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 Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.     

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.     

Effect of salinity stress on growth,peroxidase and IAA oxidase activities in vigna seedlings

The present work was carried out with the aim of studying the effect of salinity stress on growth and IAA oxidizing system (i.e. peroxidase and IAA oxidase) in vigna (Vigna unguiculata L.) seedlings. The seedlings were treated with two concentrations of sodium chloride (NaCl) 0.1 M and 0.25 M. Length, fresh and dry weight were the parameters considered for growth. Salinity effect was distinct in fresh weight and dry weight of different organs. Peroxidase and IAA oxidase activities were measured at different time intervals for both cytoplasmic and wall bound fractions. Peroxidase activity was maximum at higher stress conditions bringing about the hypocotyl growth restriction. Thus there was a clear inverse correlation between elongation and peroxidase activity. IAA oxidase activity also showed a similar trend for both cytoplasmic and wall bound fractions. The role of IAA oxidizing system in defense mechanism in response to salinity stress is discussed.     

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.     

Induction of Isoperoxidases in Resistant and Susceptible Tomato Cultivars by Meloidogyne incognita

Isoperoxidases were detected in resistant Rossol and susceptible Roma VF tomato roots uninfected and infected by Meloidogyne incognita. Syringaldazine, guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), and indoleacetic acid (IAA) were used as substrates, and the corresponding peroxidative activities were detected either in cytoplasmic or in cell wall fractions, except for IAA oxidase, which was measured in soluble and microsomal fractions. Isoperoxidase activities and cellular locations were induced differently in resistant and susceptible cultivars by nematodes. Nematode infestation markedly enhanced syringaldazine oxidase activity in cell walls of the resistant cultivar. This isoperoxidase is involved in the last step of lignin deposition in plants. Conversely, the susceptible cultivar reacted to M. incognita infection with an increase in cytoplasmic PPD-PC oxidase activity, which presumedly is involved in ethylene production; no changes in cell wall isoperoxidases were observed. IAA oxidase was inhibited in susceptible plants after nematode inoculation, whereas in resistant plants this activity increased in the soluble fraction and decreased in the microsomal fraction.     

Changes in peroxidase and IAA oxidase activities during cell elongation in Phaseolus hypocotyls

Cytoplasmic and salt-extracted peroxidase and IAA oxidase activities were studied in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA, 200 μM), naphthyl acetic acid (NAA, 100 μM) and distilled water control (DW). Peroxidase activity was assayed with four hydrogen donors during the initial phase of hypocotyl elongation. Though peroxidase activity showed a decreasing trend with time in all the hydrogen donors studied; considerable variation with different hydrogen donors was observed. NAA had maximum peroxidase activity as compared to DW or GA treatment. The activity showed a clear inverse correlation with hypocotyl growth. IAA oxidase activity showed a similar trend with growth as peroxidase activity. A highly significant correlation was observed between peroxidase and IAA oxidase activities and high molecular weight xyloglucan content (P<0.001). Finally, the possible role of peroxidase and IAA oxidase activities in hypocotyl elongation growth is discussed.     

甘露醇和6—BA处理对水稻细胞过氧化物酶及IAA氧化酶活性的影响

研究了甘露醇和６０ＢＡ处理对水稻服浮细胞再分化、过氧化物酶及ＩＡＡ氧化酶的影响。结果表明,甘露醇处理能延迟水稻细胞衰老,提高细胞再分化能力,降低细胞过氧化物酶和ＩＡＡ氧化酶活性,６－ＢＡ（２ｍｇ／Ｌ）虽然明显降低细胞过氧化物酶活性,但对ＩＡＡ氧化酶及细胞衰老无明显影响,讨论了过氧化物酶及ＩＡＡ氧化酶在水稻胚性细胞形成上的可能作用。     

Indolylacetic acid oxidase in dormant apple embryos

The IAA oxidase activity was studied during the culture of dormant apple embryos. The effect of different factors on this enzyme activity was investigated either by adding them to the reaction mixture or to the culture medium. Phloridzin was found to be the best phenolic cofactor. The development of IAA oxidase activity was stimulated by phloridzin and GA₃. The properties of apple embryos IAA oxidase allow to postulate the presence of two enzyme systems able to oxidize IAA in the material studied. The involvement of peroxidase activity in IAA oxidation was also investigated. The differences in the changes of peroxidase and IAA oxidase activities during the culture of dormant apple embryos do not permit to consider the activity of peroxidases to be identical with that of IAA oxidase.     

Changes in Paramagnetic Manganese (Mn2+) and Related Enzyme Activities in Isolated Cucumber Cotyledons During Growth: II. EFFECT OF 6-FURFURYLAMINOPURINE

Changes in the activities of IAA oxidase, peroxidase, ascorbicacid utilization (AAU), and in the level of paramagnetic manganese(Mn²⁺) have been studied during kinetin-induced growth of theisolated cucumber cotyledons in light or in dark. In kinetin-treatedcotyledons exposed to light, inhibition in the level of paramagneticmanganese corresponds with an enhancement in IAA oxidase activity.The level of paramagnetic manganese shows an inverse correlationwith IAA oxidase activity. In darkness the level of Mn²⁺ doesnot show the same correlation with IAA oxidase activity as inthe light. Kinetin stimulates peroxidase activity both in thelight and in darkness. Enhancement of IAA oxidase activity andno corresponding change in the level of paramagnetic manganeseindicates that the oxidation of IAA in dark-grown, kinetin-treatedcotyledons is brought about by peroxidase. It appears that thephenolic cofactors required for the oxidation of manganese andIAA may be limiting in kinetin-treated cotyledons in darkness.Thus in the light, IAA oxidation seems to be brought about byperoxidase as well as manganese, whereas in darkness it is mediatedby peroxidase alone. Increase in IAA oxidase activity duringkinetin-induced growth of the isolated cotyledons is incompatiblewith the idea that increased IAA oxidase activity would limitthe availability of auxin for growth. Kinetin does not mimicthe action of light on IAA oxidase activity; on the contrary,it removes the inhibitory effect of light on IAA oxidase activityprobably through the synthesis of IAA oxidase activators.     

Effect of dicarboxylic acids on the peroxidase-IAA oxidase isozymes of soybean callus

Multiple forms of peroxidase with indole-3-acetic acid (IAA) oxidase activity were detected in callus cultures from soybean seeds [ Glycinc max (L.) Merrill, cv. Acme] using ion-exchange chromatography and polyacrylamide gel electrophoresis. The properties of the IAA oxidase were studied with a partially purified fraction eluted from a DEAE cellulose column. At pH 5.7. p-coumaric acid and MnCl₂ were required as cofactors and H₂O₂ was not able to replace them, but H₂O₂ eliminated the usual lag period of the reaction. Activation effects obtained with some dicarboxylic acids acting only on IAA oxidase are shown. These effects were studied at different pH values and oxalic acid was found to be the most efficient activator, particularly at pH 4.6. Activation by oxalic acid occurred even in the absence of MnCl₂, but the presence of this salt produced a synergistic effect. IAA oxidase showed a sigmoidal kinetic behaviour at pH 5.7 changing to hyperbolic at pH 4.6     

Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

IAA Oxidase preparations from fresh and aged Ipomoea batatas tuber discs

IAA oxidase preparations from fresh sweet potato tuber discs oxidized IAA only in the presence of added phenolic cofactors, and the pH optimum for enzyme activity depended on the cofactor used. Ageing of tuber discs, either by aeration in distilled water or by incubation on moist filter paper, resulted in increased peroxidase and phenol-stimulated IAA oxidase activities, as well as the development of IAA oxidase activity in the absence of added cofactors. High phenolase activity of fresh tuber discs decreased considerably with ageing. Phenol-stimulated IAA oxidase activity reached maximal levels before IAA oxidase activity in the absence of added cofactors. Enzyme preparations from aged tuber discs had double pH optima, similar to those previously described for sweet potato root IAA oxidase preparations. IAA in the concentration range 10^?4 to 10^?2 M inhibited the increase in peroxidase and IAA oxidase activities with ageing. DCP-stimulated IAA oxidase activities in preparations from both fresh and aged sweet potato tuber discs were inhibited by manganous ion.     

Peroxide Levels and the Activities of Catalase, Peroxidase, and Indoleacetic Acid Oxidase during and after Chilling Cucumber Seedlings

The activities of catalase, peroxidase, indoleacetic acid (IAA) oxidase and peroxide levels in cucumber plants during and after chilling were determined. During 96 hours at 5 C and 85% relative humidity, catalase activity declined, IAA oxidase activity increased, and peroxide concentrations increased. Peroxidase activity was not affected by chilling. When chilled plants were returned to 25 C to recover, enzyme activities and peroxide concentration were restored to their prechilling levels. The increase in peroxide and IAA oxidase activity may inactivate or destroy IAA and thus retard growth.     

Studies on auxin protector substances, IAA-oxidase and peroxidase in cotyledons of Pharbitis nil

The nature of macromolecular "auxin protector substances" causinglag periods rather than inhibition in the rate of IAA oxidationwas reinvestigated. Three different peaks were separated bySephadex gel filtration; each was then examined by means ofenzymatic (IAA oxidase, peroxidase) and electrophoretic techniquesand correlated with the activities of both enzymes and withzymogram patters. The auxin protector activity of the high molecularweight fractions increased after high temperature treatment.On the basis of experiments involving dialysis and chromatographybefore and after heating, auxin protectors appear to be complexesof macromolecules with small molecules. (Received May 18, 1971; )     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Cloning and characterisation of a basic IAA oxidase associated with root induction in Vitis vinifera.

Changes in apoplastic peroxidases during auxin-induced in vitro rooting of cultured grapevine (Vitis vinifera L. cv. Touriga) stems have been studied. The largest increase in peroxidase activity (EC 1.11.1.7) was associated with the early stages of root initiation and could be attributed to an increase in activity of an apoplastic 36 kDa cationic peroxidase (PxB2). Relative to other peroxidases, PxB2 demonstrated high indole-3-acetic acid (IAA) oxidase activity and apparently contributed the majority of potential IAA oxidase activity in rooting tissues. The distribution of this peroxidase in developing roots additionally associates it with early phases of growth restriction. PxB2 was purified from cell wall extracts prepared from the basal 1 cm of rooting stems. Microsequencing and subsequent cloning of its corresponding 3' truncated cDNA (encoding 255 amino acids of the mature protein) revealed it to have a typical class III peroxidase structure. The results suggest that this class III peroxidase with IAA oxidase activity is important for the control of IAA levels during root initiation and development.     

乙烯利对甘蔗分蘖期茎三种酶活性的影响及其与分蘖的关系

在分蘖前期喷施适宜浓度(100mg/L)乙烯利提高了蔗茎的过氧化物酶活性和IAA氧化酶的活性。用100mg/L乙烯利处理后,两个品种根部的过氧化物酶和IAA氧化酶活性明显高于上部的活性,并且比对照和400mg/L乙烯利处理的效果明显。乙烯利处理后新台糖16号上部节间的酸性转化酶活性始终高于下部节间的酶活性,其中100mg/L乙烯利处理下部节间的明显低于对照的;乙烯利处理后新台糖22号茎内的酸性转化酶活性也低于对照的,但与对照的差异相对比新台糖16号的小。     

Kinetic properties of IAA oxidase from mung bean cotyledons

A crude enzyme preparation from mung bean cotyledons was separated into peroxidative and non-peroxidative IAA oxidase on a DEAE-cellulose column. Both fractions differed in their pH optima, K_m and V_max. The K_m and V_max of non-peroxidative IAA oxidase were higher than those of peroxidative IAA oxidase. Peroxidative IAA oxidase showed a linear increase in absorption at 247 and 254 nm after a short lag of 2–3 min. The addition of catalytic amounts of hydrogen peroxide eliminated the lag period and also enhanced the rate of IAA degradation. The non-peroxidative IAA oxidase fraction, however, did not exhibit any significant increase in absorption at 247 and 254 nm and showed a lag period of 5 min which was not affected by hydrogen peroxide. Instead, addition of the same catalytic amount of hydrogen peroxide inhibited the rate of IAA degradation. The peroxidative IAA oxidase fraction exhibited the reaction kinetics characteristic of peroxidase-catalysed IAA degradation. The rate of IAA oxidation by purified non-peroxidative IAA oxidase was very low. The slow rate of catalysis shown by non-peroxidative IAA oxidase appears to be due to the presence of inhibitor(s).