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.     

Effect of Indoleacetic Acid and Related Indoles on Lactobacillus sp. Strain 11201 Growth, Indoleacetic Acid Catabolism, and 3-Methylindole Formation

A study was conducted to determine the activity of the 3-methylindole (3MI)-forming enzyme in Lactobacillus sp. strain 11201. Cells were incubated anaerobically with 17 different indolic and aromatic compounds. Indoleacetic acid (IAA), 5-hydroxyindoleacetic acid, 5-methoxy-3-indoleacetic acid, indole-3-pyruvate, or indole-3-propionic acid induced 3MI-forming activity. The highest total enzyme activity induced by IAA was observed in cells incubated with an initial concentration of 1.14 mM IAA. Peak activity of the 3MI-forming enzyme occurred 4 h after bacteria were incubated with either 0.114 or 1.14 mM IAA. Enzyme activity peaked earlier (2 h) and disappeared more rapidly at 5.7 mM IAA than at other concentrations of IAA. The effects of IAA and 3MI on the growth of Lactobacillus sp. strain 11201 and formation of 3MI from IAA also were determined. Bacterial growth and 3MI formation from IAA were reduced in medium containing exogenous 3MI. The growth depression observed in medium containing 5.7 mM IAA appears to be due to the toxicity of 3MI rather than IAA. The formation of 3MI in this ruminal Lactobacillus sp. is mediated by an inducible enzyme, and as 3MI accumulates, bacterial growth and rates of 3MI formation from IAA are reduced.     

Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by Pseudomonas putida Strain 1290

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.     

Indole-3-acetic acid production,solubilization of insoluble metal minerals and metal tolerance of some sclerodermatoid fungi collected from northern Thailand

Sclerodermatoid fungi basidiomes were collected from northern Thailand and pure cultures were isolated. The morphology and molecular characteristics identified them as Astraeus odoratus, Phlebopus portentosus, Pisolithus albus and Scleroderma sinnamariense. This study investigated the in vitro ability of selected fungi to produce indole-3-acetic acid (IAA), to solubilize different toxic metal (Co, Cd, Cu, Pb, Zn)-containing minerals, and metal tolerance. The results indicated that all fungi are able to produce IAA in liquid medium. The optimum temperature for IAA production of all fungi was 30 °C, and the optimum concentration of L-tryptophan of Astraeus odoratus, Pisolithus albus and Scleroderma sinnamariense was 2 mg ml^?1. The highest IAA yield (65.29?±?1.17 μg ml^?1) was obtained from Phlebopus portentosus after 40 days of cultivation in culture medium supplemented with 4 mg ml^?1 of L-tryptophan. The biological activity tests of fungal IAA showed that it can simulate coleoptile elongation, and increase seed germination and root length of tested plants. In addition, the metal tolerance and solubilizing activities varied for different minerals and fungal species. The presence of metal minerals affected fungal growth, and cobalt carbonate showed the highest toxicity. The solubilization index decreased when the concentration of metal minerals increased. Astraeus odoratus showed the lowest tolerance to metals. This is the first report of in vitro IAA production, solubilization of insoluble metal minerals and metal tolerance abilities of the tested fungi.     

Studies on the oxidation of indole-3-acetic Acid by peroxidase enzymes. I. Colorimetric determination of indole-3-acetic Acid oxidation products

The method described here is based on a brief report by Harley-Mason and Archer. It involves the use of p-dimethylaminocinnamaldehyde (DMACA), a vinylogue of Ehrlich's reagent, as a color reagent for indoles. Colorimetric analyses of indoleacetic acid (IAA) oxidation reaction mixtures were made with the DMACA reagent as a solution rather than a spray. DMACA reagent will yield a wine-red color with IAA oxidation products in solution. Under similar conditions DMACA reacts with authentic IAA to yield only slight coloration at best. In comparison with other indoles, DMACA is more relative with IAA oxidation reaction products than either Salkowski or Ehrlich's reagents. Data discussed support a concept that the color produced with DMACA is due to the presence of tautomeric oxidation product(s) of IAA.     

Metabolism of ingested auxins in the bug Lygus disponsi: Conversion of indole-3-acetic acid and gibberellin

Metabolism of indole-3-acetic acid (IAA) and gibberellic acid (GA) in the gut of the bug Lygus disponsi was investigated. IAA was converted to some IAA metabolites with auxin activity in vivo but not in vitro. They were ninhydrin and anilinehydrogenphthalate negative. GA was not converted in vivo. By means of Avena straight growth test auxin activity was not detected in either the salivary gland of IAA-feeding bugs nor in the salivary gland of GA-feeding bugs. The significance of IAA conversion in the gut of L. disponsi is discussed.     

Isolation and characterization of wheat peroxidase isoenzyme B1

Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn²⁺ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H₂O₂ from oxygen. Mn²⁺ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.     

Auxin production by the Klebsiella planticola strain TSKhA-91 and its effect on development of cucumber (Cucumis sativus L.) seeds

Capacity of Klebsiella planticola strain TSKhA-91 for synthesis of indole-3-acetic acid (IAA) and other auxins was studied. The qualitative and quantitative composition of these compounds depends on the presence of exogenous tryptophan and on the nitrogen source. The highest IAA yield was obtained at the stationary phase of growth. Addition of L-tryptophan to the medium resulted in a significant increase (up to 85.5 μg/mL) of auxin biosynthesis, especially in the presence of nitrates. Thin-layer chromatography revealed that the indole-3-acetamide pathway was not active in this strain. The biological activity of auxins was confirmed by assay with kidney bean cuttings; the height of root formation and root number increased 16- and 6-fold, respectively. Under conditions of low-temperature stress, protective effect of K. planticola TSKhA-91 on development of cucumber (Cucumis sativus L.) seeds and stimulation of germination and root formation by its seeds were shown.     

Differentiation between indoleacetic Acid and the citrus auxin by column chromatography

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.     

Early effects of boron deficiency on indoleacetic Acid oxidase levels of squash root tips

The indoleacetic acid (IAA) oxidase activity of root tips of boron-sufficient, -deficient, recovering, and IAA-treated boron-sufficient squash plants (Cucurbita pepo L.) was determined. Apical and subapical root sections displayed an increase in IAA oxidase activity between 6 and 9 hours after boron was withheld, and after 24 hours the activity of the apical sections showed a 20-fold increase over +B controls. Root elongation of -B plants was inhibited before an increase in oxidase activity could be detected. Roots of plants subjected to 12 hours of -B treatment and then transferred to +B treatment for recovery regained normal elongation rates and oxidase activity within 18 to 20 hours. IAA treatment of +B plants increased IAA oxidase activity of apical and subapical root sections and also inhibited root elongation and caused symptoms similar to -B treatments.     

The effect of natural and synthetic auxins on the growth,metabolite content and antioxidant response of green alga Chlorella vulgaris (Trebouxiophyceae)

The effect of exogenously applied natural [indole-3-acetic acid (IAA), phenylacetic acid (PAA), indole-3-butyric acid (IBA)] and synthetic [1-naphthaleneacetic acid (NAA)] auxins on the growth and metabolism of green microalga Chlorella vulgaris was examined. Exogenous auxins acted in a concentration-dependent manner on algal growth. Phytohormones at concentration of 100 μM inhibited algal growth expressed as the number of cells. IAA and IBA displayed the highest biological activity at 0.1 μM, whereas PAA and NAA were characterized by the greatest stimulatory effect on the number of cells at 1 μM. Treatment with IAA and IBA at 0.1 μM or NAA and PAA at 1 μM increased the concentration of photosynthetic pigments, monosaccharides and soluble proteins in C. vulgaris. Moreover, all auxins stimulated enzymatic (ascorbate peroxidase, catalase, superoxide dismutase) and non-enzymatic antioxidant (ascorbate, glutathione) systems in C. vulgaris, and therefore, suppressed lipid peroxidation and hydrogen peroxide accumulation. The data supports the hypothesis that auxins play a central role in the regulation of C. vulgaris growth and metabolism and the components of cellular redox systems that are thought to have a prominent role in the regulation of auxin-dependent processes.     

Catabolic Pathway for the Production of Skatole and Indoleacetic Acid by the Acetogen Clostridium drakei, Clostridium scatologenes, and Swine Manure

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.     

Hormonal Relations in the Phototropic Response IV. Light-Induced Changes of Endogenous Auxins in the Coleoptile

Beside indoleacetic acid (IAA), 3 auxins were found by chromatographic resolution of acidic fractions of Avena and Zea coleoptile tips. One of these auxins, designated P, occurred at levels of activity approaching those of IAA. The other 2 auxins, termed F and M, occurred at lower levels of activity. When the auxins of the excised coleoptile tips were isolated immediately after equilateral or unilateral irradiation with blue light at first positive energies, the ratio of IAA to the other auxins increases. This rise is the result of a decrease in P and F, and probably an increase in IAA. Light did not affect materially the total auxin content. It is suggested that P and F might be associated with the basipetal transport inequalities of IAA in phototropism.

P has been partially characterized. Its R_F on chromatograms developed in ammoniacal isopropanol is about 0.65. It is converted to IAA in vitro by heat. The ultraviolet absorption spectrum of chromatographically resolved P also suggests an indolyl complex. P is not readily transported basipetally, and the slope of its relative concentration-response curve (Avena section test) is lower than that of IAA. P does not appear to be any of the chemically characterized native auxins.

     

Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by MicroRNA847 Upregulates Auxin Signaling to Modulate Cell Proliferation and Lateral Organ Growth in Arabidopsis

MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression.     

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.