A fluorometric method for the kinetic assay of indole-3-acetic acid oxidase.

The oxidation of IAA by peroxidase (1) and by more specific oxidases (2) leads to the formation of products which may have physiological activity (3, 4). The colorimetric estimation of IAA oxidation products involving reaction with p-dimethylaminocinnamaldehyde (DMACA) is reported to be more sensitive than other end point determinations such as the Salkowski and Ehrlich procedures which monitor the disappearance of IAA (5). These methods are end point procedures and, as such, are awkward and time consuming and present difficulties in obtaining kinetic data and measuring lag times. IAA oxidation has also been monitored by measuring ¹⁴CO₂ released from [1-¹⁴C] IAA (6) and uv spectral shifts during oxidation of IAA were reported by Meudt (3). The present paper reports a new procedure for the assay of horseradish peroxidase catalyzed oxidation of IAA. The assay procedure is based on the continuous measurement of a fluorescent product of the reaction.     

Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls

The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3-methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3-acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.     

Oxidation of indole-3-acetic Acid-amino Acid conjugates by horseradish peroxidase

The stability of 21 amino acid conjugates of indole-3-acetic acid (IAA) toward horseradish peroxidase (HRP) was studied. The IAA conjugates of Arg, Ile, Leu, Tyr, and Val were oxidized readily by peroxidase. Those of Ala, β-Ala, Asp, Cys, Gln, Glu, Gly, and Lys were not degraded and their recovery was above 92% after 1 hour incubation with HRP. A correlation between the stability of IAA conjugates toward peroxidase-catalyzed oxidation and the hydrophobicity of the amino acid moiety conjugated to IAA was demonstrated. Polar amino acid conjugates of IAA are more resistant to HRP-catalyzed oxidation.     

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues

Exogenous [¹⁴C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [¹⁴C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Indole-3-acetic acid and indole-3-ethanol in light-grown Pisum sativum seedlings and their localization in chloroplast fractions

Studies have been carried out on the compartmentation ofindole-3-acetic acid (IAA) and related indoles in Pisum sativum cv. Meteor. By the use of HPLC, GC and combined GC-MS, data were obtained demonstrating the presence of IAA and indole-3-ethanol (IEt) in light-grown pea seedlings. HPLC, GC and GC-MS analyses also confirmed IAA as an endogenous constituent in pea chloroplast fractions while HPLC and GC provided strong evidence for the presence of IEt in chloroplast preparations.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Esters of indole-3-acetic Acid from Avena seeds

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK_a values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.     

Destruction of indole-3-acetic acid during the aerobic oxidation of sulfite

Indole-3-acetic acid (IAA) was rapidly destroyed in the presence of Mn²⁺, oxygen and sulfite ion. The optimal pH for the reaction was between 5 and 6. The destruction was dependent on the aerobic oxidation of sulfite, but was not inhibited by superoxide dismutase. Tracer studies indicate that IAA was converted into at least 3 compounds. Decarboxylation of IAA was not involved in the destruction.     

Bistability and reaction thresholds in the phenol-inhibited peroxidase-catalyzed oxidation of indole-3-acetic acid

A model mechanism for the phenol-inhibited peroxidase-catalyzed oxidation of indole-3-acetic acid (IAA) is proposed and analyzed. The model involves an autocatalytic free radical species that sustains IAA oxidation and the phenolic inhibitor acting as a free radical scavenger. Under a fixed set of parameter values, the model exhibits a coexistence of two stable steady states. This bistability phenomenon explains the origin of the experimentally observed threshold inhibitor concentrations above which IAA oxidation stops. The variation of the inhibitor threshold level with enzyme and substrate concentrations are reproduced by the model almost quantitatively.     

Activation of indole-3-acetic acid oxidase from horseradish and Prunus by phenols and H2O2

The enzyme-catalysed oxidation of indole-3-acetic acid (IAA) was sytematically investigated with respect to enzyme source and cofactor influence using differential spectrophotometry and oxygen uptake measurement. Commercially-available horseradish peroxidase (HRP) and a peroxidase preparation from Prunus phloem showed identical catalytic properties in degrading IAA. There was no lag phase of IAA oxidation with any of the reaction mixtures tested. Monophenols exhibited a much stronger stimulatory effect than inorganic cofactors, but during the incubation of IAA the phenols were also gradually oxidised. Hydrogen peroxide (H₂O₂) in combination with monophenols accelerated peroxidation of the monophenol and IAA oxidation simutaneously. Since photometric determination of IAA was affected by oxidation products of dichlorophenol or phenol contamination of the enzyme preparation used, the standard IAA absorption measurements appear to be susceptible to methodological errors. Under certain incubation conditions a catalase-like activity of HRP during the course of IAA oxidation was noted and substrate inhibition was observed above 1.5 × 10^\s-4 M IAA. Some concepts concerning the mode of activation of the enzyme-catalysed IAA oxidation are deduced from the experimental results.     

Morphogenic responses of debudded tobacco plants to gibberellic Acid and indole-3-acetic Acid

Stem applications of indole-3-acetic acid (IAA) or gibberellic acid (GA) did not prevent or alter tumor or teratoma formation in debudded tobacco plants (Nicotiana tabacum L., var. One Sucker). The materials produced intense (in case of GA) and moderate (in case of IAA) stem proliferations when applied to debudded plants but were without effect on intact plants.

The results suggest that debudding-tumors are probably not related to or a result of an auxin or gibberellin deficit and that total debudding has a marked physiological effect on the plant. The altered physiological condition of the debudded plant, indicated by its responses to IAA and GA, may likely be related to tumor and teratoma formation.

     

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.     

Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers

A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA.     

The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation

Indole-3-acetic acid (IAA) was oxidized by horseradish peroxidase, but ester and amide conjugates of IAA were not degraded. Addition of indoleacetyl-myo-inositol, indoleacetyl-L-aspartate, indoleacetylglycine, indoleacetyl-L-alanine, indoleacetyl-D-alanine, or indoleacetyl--alanine did not affect the rate of oxidation of IAA by horseradish peroxidase. Peroxidase preparations from Pisum sativum L. and Zea mays L. behaved similarly in that they rapidly oxidized IAA, but not conjugates found in the plant from which the peroxidase was prepared. These results indicate that conjugation could affect the stability of IAA in vivo.Abbreviation IAA Indole-3-acetic acid     

Nutrient salts promote light-induced degradation of indole-3-acetic Acid in tissue culture media

The disappearance of indole-3-acetic acid (IAA) from cell-free liquid culture medium was followed in response to nutrient salts found in Murashige-Skoog salt base, light, and pH range of 4 to 7. The loss of IAA was accelerated by light or Murashige-Skoog salts. However, the combination of both light and Murashige-Skoog salts acted synergistically to catalyze the destruction of over 80% of the original IAA within 7 days of continuous incubation. Under these same conditions, the loss of IAA was decreased to approximately 50% by adjusting the initial pH of the medium to 7. Iron was identified as the single major contributor to light-catalyzed destruction of IAA. Removal of nitrates, which represented 87% of the molar salt composition, also reduced the light-catalyzed loss of IAA. Treatments that protected IAA from degradation, such as darkness or removal of iron from the medium, suppressed the growth of muskmelon (Cucumis melo. Naud., var. reticulatus) callus tissue cultured for 30 days. Treatments in the light that rapidly degraded IAA resulted in maximum growth. Consequently, the brief exposure to IAA prior to degradation was apparently sufficient to initiate physiological changes required for growth. Possible approaches to the preservation of IAA during incubation are discussed.     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

Inhibitory oxidation products of indole-3-acetic Acid: 3-hydroxymethyloxindole and 3-methyleneoxindole as plant metabolites

Extracts of pea seedlings (Pisum sativum, variety Alaska) oxidize indole-3-acetic acid to a bacteriostatic compound which has been identified as 3-hydroxymethyloxindole. At physiological pH this compound is readily dehydrated to 3-methyleneoxindole, another bacteriostatic agent. The extracts of pea seedlings also contain a reduced triphosphopyridine nucleotide-linked enzyme which reduces 3-methyleneoxindole to 3-methyloxindole, a non-toxic compound.

These enzymatic reactions also take place in intact seedlings; thus, a pathway of indole-3-acetic acid degradation via oxindoles appears to be pertinent to plant metabolism.

The significance of such metabolism lies in the fact that a key intermediate of this pathway, 3-methyleneoxindole, is a sulfhydryl reagent capable of profound effects on metabolism and growth.