Isolation of Indole-3-ethanol Oxidase from Cucumber Seedlings

Previous work in this laboratory has shown that cucumber (Cucumis sativus L.) seedlings contain large amounts, relative to other indolic compounds, of extractable indole-3-ethanol (IEt); tracer studies have established that IEt is metabolized to IAA. We have now succeeded in isolating an enzyme from these seedlings which catalyzes the oxidation of IEt to indole-3-acetaldehyde (IAAld). The identification of the product as IAAld was based on solvent partitioning of the free aldehyde and its bisulfite adduct and radiochromatography following incubation of enzyme with ¹⁴C-IEt. A novel, quantitative colorimetric test for IAAld was also developed utilizing the Salkowski reagent. Partial purification of the enzyme was achieved by salt gradient chromatography on Bio-Rex 70, heating the preparation to 70 C, and chromatography on Sephadex G-150. This purification procedure yielded an enzyme activity purified in excess of 3000-fold, and studies on a standardized Sephadex column suggest a molecular weight of the enzyme of approximately 105,000. The reaction was found to proceed only aerobically; and, in the absence of other electron acceptors, O₂ appears to be reduced to H₂O₂. The enzyme has nearly maximum activity from pH 8 to 11.     

Metabolism of indole-3-acetaldoxime in plants

Summary Living tissues of diverse plants representing 17 families were infiltrated with indole-3-acetaldoxime (IAAld oxime) in phosphate buffer, pH 6, and incubated for 3 hours at 25°C. Indole compounds were then extracted, separated and identified by paper or thin-layer chromatography (TLC). Indole-3-acetic acid (IAA) was quantitatively determined. Every tissue tested converted the oxime to IAA and tryptophol (T-ol). While accumulation of indole-3-acetonitrile (IAN) was observed in the non-acidic fractions of extracts of tissues of 8 species, indole-3-acetaldehyde (IAAld) accumulated in only a single tissue viz. Amaranthus shoot.IAAld oxime undergoes spontaneous hydrolysis at pH values below 4.7 leading to the formation of IAAld. Ce l-free preparations of etiolated Avena coleoptiles appear to contain an enzyme system capable of hydrolysing the oxime to IAAld. In the presence of such preparations, more IAAld and IAA are formed at all tested durations than the spontaneously formed IAAld. In the presence of bisulfite or semicarbazide, no IAA is formed, suggesting the intermediary formation of IAAld. The compound trapped with sodium bisulfite resembles very closely synthetic IAAld in its IR spectrum.In intact tissues, therefore, IAAld oxime appears to be first hydrolysed to IAAld which is then partly oxidized to IAA and mostly reduced to T-ol. Besides other evidence, formation of T-ol in every instance is believed to indicate the intermediary formation of IAAld. The nitrile pathway is considered to be only of minor importance in normal IAA biogenesis in the majority of higher plants.     

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step “IAAld → IAA” from a silk-gland extract of Bombyx mori. The enzyme, designated “BmIAO1”, contains two 2Fe–2S iron–sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp.

Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC–MS/MS: liquid chromatography–tandem mass spectrometry; Trp: tryptophan     

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.     

La biosynthèse de l'acide indolyl-3-acétique en liaison avec le métabolisme du tryptophol et de l'indolyl-3-acétaldéhyde chez Rhizobium

Several indolic compounds are formed when tryptophan or tryptamine is metabolized by Rhizobium. Among these are indole-3-acetaldehyde (IAAld), tryptophol (Tr-ol), and indole-3-acetic acid (IAA). The metabolic relationship among the three compounds was investigated. The experiments were carried out either in the culture medium of growing Rhizobium cultures or in suspensions of washed bacterial cells. In neither case Tr-ol would function as a precursor of IAA, but tryptophan-2-¹⁴G gave rise to the formation of both IAA and Tr-ol. The ratio of IAA to Tr-ol depended on the experimental conditions, shaking favoring the formation of IAA. Also IAAld gave rise to the formation of IAA and Tr-ol when incubated with suspensions of washed cells. The ratio of the two compounds depended on experimental conditions such as pH value and shaking, the latter reducing the formation of Tr-ol. These results cannot be explained by the assumption of a dismutation mechanism catalyzed by a single enzymatic unit. The operation of two enzyme systems, responsible for the reduction and the oxidation, respectively, of IAAld is suggested and discussed.     

Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae

Summary Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from l-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.     

Identification of indole-3-acetaldehyde and indole-3-acetaldehyde reductase in Chinese cabbage

Indole-3-acetaldehyde (IAAId) was identified as a natural compound in Chinese cabbage ( Brassica campestris L. ssp. pekinensis cv. Granat) seedlings by chemical conversion to indole-3-acetaldoxime (1AOX) followed by mass spectroscopy. The lAAId reductase (EC 1.2. 3.1), an enzyme with a molecular mass of 32 kDa, was extracted, purified 5-fold and characterized. The enzymatic IAAld reduction showed a pH optimum at 6–7 and a marked preference for NADPH as cofactor The K_m value for IAAld was 125 μ M , for NADPH 36 μ M . The enzyme reaction was inhibited at high NADPH concentrations (>200 μ M ) and modulated by IAA and indole-3-ethanol (IEt). Sulfhydryl reagents inhibited IEt formation, suggesting the participation of SH-groups in the reaction. Phenylacetaldehyde and benzaldehyde were competitive substrates, while acetaldehyde acted partly as an inhibitor, and partly as an activator on the IAAld reduction. IAAld reductase activity was also detected in other Brassica species. The importance of this enzyme is discussed with respect to the possibilities of IAA biosynthesis in the Brassicaceae.     

Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.     

Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut / huitlacoche)

Production of indole-3-acetic acid (IAA) by four strains of the maize pathogen Ustilago maydis was analyzed. The fungus induces gall formation on its host plant and IAA production by  U. maydis may be required as a pathogenicity or virulence factor. The study included the FB2 wild-type strain and the 103, 130FZ and 130FT mutants. Results show that treatment with clofibric acid, alone or in combination with UV light, can be used to obtain  U. maydis strains with defective IAA production in vitro, as quantified with the Salkowski reagent and by HPLC. The strain with the lowest production was 130FT, and its peak IAA level represented only 16% of the highest value obtained for the FB2 wild-type strain (124 μg/ml). Received: 11 April 1996 / Received last revision: 5 September 1997 / Accepted: 11 September 1997     

An in Vitro System of Indole-3-Acetic Acid Formation from Tryptophan in Maize (Zea mays) Coleoptile Extracts

The formation of a product from tryptophan that had the same retention time as that of authentic indole-3-acetic acid (IAA) on high performance liquid chromatography was detected in crude extracts of maize (Zea mays) coleoptiles. The product was identified as IAA by mass spectrometry. The IAA-forming activity was co-purified with an indole-3-acetaldehyde (IAAld) oxidase activity by chromatography on hydrophobic and gel filtration (GPC-100) columns. During purification, the IAA-forming activity, rather than that of IAAld oxidase, decreased; but when hemoprotein obtained from the same tissue was added, activity recovered to the same level as that of IAAld oxidase. The promotive activity of the hemoprotein was confirmed by the result that the activity coincided with amounts of the hemoprotein after GPC-100 column chromatography. The hemoprotein was characterized and identified as a cytosolic ascorbate peroxidase (T. Koshiba [1993] Plant Cell Physiol [in press]). The reaction of the IAA-forming activity was apparently one step from tryptophan. The activity was inhibited by 2-mercaptoethanol. The optimum temperature for the IAA-forming system as well as for the IAAld oxidase was 50 to 60[deg]C, and the acitivity at 30[deg]C was one-third to one-half of that at 60[deg]C. The system did not discriminate the L- and D-enantiomers of tryptophan.     

The formation of tryptophol glucoside in the tryptamine metabolism of pea seedlings

Summary Tryptamine was converted by etiolated pea seedlings into IAA, tryptophol, and an appreciable amount of an unknown metabolite. This latter compound was characterised by TLC and electrophoresis and identified, by mass spectrometry and enzymatic cleavage, as tryptophol glycoside: indole-3-ethyl--d-glycopyranoside.Abbreviations IAA indole-3-acetic acid - IAAld indole-3-acetaldehyde - TOH tryptophol - TO-glc tryptophol glucoside     

IAA-synthase, an enzyme complex from Arabidopsis thaliana catalyzing the formation of indole-3-acetic acid from (S)-tryptophan

An enzyme complex was isolated from Arabidopsis thaliana that catalyzes the entire pathway of biosynthesis of the major plant growth hormone, indole-3-acetic acid (IAA), from (S)-tryptophan. The 160-180 kDa, soluble complex catalyzes a strictly O2-dependent reaction which requires no further added factors and is stereospecific for the substrate (S)-tryptophan (app. Km = 120 microM). H2(18)O labeling proved that both oxygen atoms of IAA were delivered via H2O. This, as well as immunological evidence for the presence of a nitrilase-like protein in the complex, suggests the reaction to proceed via the intermediate indole-3-acetonitrile. IAA-synthase forms a tight metabolite channel committed to IAA production and occurs in shoots, roots and cell cultures of A. thaliana.     

Effect of Lanthanum on Rooting of In Vitro Regenerated Shoots of Saussurea involucrata Kar. et Kir

In present study, the effect of lanthanum (La) on the rooting of regenerated shoots of Saussurea involucrata Kar. et Kir was analyzed. Rooting occurred from regenerated shoots inoculated on a medium supplemented with La, the plant rooting hormone indole-3-acetic acid (IAA), or both La and IAA together. The highest rooting efficiency (96%), root number/shoot (8.5), and root length (63 mm) were recorded in shoots cultured on medium containing 2.5 μM IAA combined with 100 μM La(3+). In order to elucidate the mechanism of rooting enhancement by La, we examined dynamic changes in antioxidant enzyme activities in plant tissue over time in culture. We found that the activities of peroxidase (POX) and superoxide dismutase (SOD) were significantly higher in plant tissue cultured in IAA plus La than in La or IAA alone. At the same time, the highest H(2)O(2) content was detected in plant tissue in the presence of 2.5 μM IAA plus 100 μM La(3+). In light of these data and previous results, we speculate that La enhanced IAA-induced rooting by acting as a mild abiotic stress to stimulate POX and SOD activities in plant cells. Then, IAA reacted with oxygen and POX to form the ternary complex enzyme-IAA-O(2) that dissociated into IAA radicals and O(2)(-). Subsequently, IAA-induced O(2)(-) readily converted to hydroxyl radical (HO·) via SOD-catalyzed dismutation. Finally, cell wall loosening and cell elongation occurred as a consequence of HO-dependent scission of wall components, leading to root growth. The treatment of IAA combined with La resulted in the highest plantlet survival (80%) compared to single treatments with IAA or La alone. These data suggest that rare earth elements enhance root morphogenesis and the growth of S. involucrata.     

A novel,simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

A soluble auxin-binding protein from mung bean hypocotyls has indole-3-acetaldehyde reductase activity

To clarify the roles of auxin-binding proteins (ABPs) in the action of auxin, soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid (2,4-D)-linked Sepharose 4B. A 39-kDa polypeptide was retained on the affinity column and eluted with a solution containing IAA or 2,4-D, but not with a solution containing benzoic acid. The protein was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 77 kDa by gel filtration and 39 kDa by SDS-PAGE. We designated this protein ABP39. The partial amino acid sequences of ABP39, obtained after chemical cleavage by CNBr, revealed high homology with alcohol dehydrogenase (ADH; EC 1.2.1.1). While the ABP39 was not capable of oxidizing ethanol, it did catalyze the reduction of indole-3-acetaldehyde (IAAld) to indole-3-ethanol (IEt) with an apparent K_m of 22 μ M. The IAAld reductase (EC 1.2.3.1) is specific for NADPH as a cofactor. The ABP39 also catalyzed the reduction of other aldehydes, such as acetaldehyde, benzaldehyde, phenylacetaldehyde and propionealdehyde. Indole-3-aldehyde was a poor substrate. The enzyme activity was inhibited by both indole-3-acetic acid and 2,4-D in a competitive manner. Therefore, the enzyme is considered to be retained on the affinity column by recognition of auxin structure.     

Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff: Characterization of the Enzyme

Indole-3-ethanol oxidase (IEt oxidase) from Phycomyces blakesleeanus Bgff.(P.b.) is a 56 kD polypeptide as determined by gel filtration. The reaction products are indole-3-acetaldehyde (IAAld) and, possibly, H₂O₂. Enzyme activity (33-45% ammonium sulfate fraction) shows a broad pH optimum and simple Michaelis-Menten kinetics (K_m 7 micromolar, Hill coefficient 0.95). Flavin adenine dinucleotide increases enzyme activity particularly under anaerobic conditions. Iodoacetate and HgCl₂ drastically inhibit the enzyme. With IAAld, product inhibition is observed at micromolar concentrations. IAA and some other acidic substituted indoles reduce enzyme activity but only at higher concentrations.     

Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

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.     

Oxindole-3-acetic Acid: Physical Properties and Lack of Influence on Growth

The auxin activity of OAA was studied. The assays used were the Avena coleoptile curvature and section tests, and the first internode test. OAA was completely inactive to these assays. Physical tests indicated no color reaction with modified Salkowski reagent (Gordon-Weber) and no activity of any of the biochromatograms run with OAA. These data indicate that OAA is inactive as an auxin due to its characteristics of structural difference from IAA and further lends some speculation as to the site of activity of IAA as an auxin.