Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition of Exogenous IAA in Arabidopsis thaliana

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D-glucopyranose, [2′,2′-²H₂]-2-oxoindole-3-acetic acid, and 1-O-([2′,2′-²H₂]-2-oxoindole-3-acetyl)-β-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Identification of oxindole-3-acetic acid,and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds

Oxindole-3-acetic acid (OxIAA) has been identified in germinating seeds of Scots pine (Pinus sylvestris) using gas chromatography-mass spectrometry. Seeds germinated for 5 d contained 2.7 ng OxIAA·g^-1 (dry weight) whereas ungerminated seeds contained 0.2 ng·g^-1. Isotopically labelled OxIAA was formed in seeds incubated with [1-¹⁴C]-, [2-¹⁴C]- or [²H₅]indole-3-acetic acid.Abbreviations DDC sodium diethyldithiocarbamate - GC gas chromatography - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - MS mass spectrometry - OxIAA oxindole-3-acetic acid - PVP polyvinylpyrrolidone - TMS trimethylsilyl     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Indole-3-methanol as an intermediate in the oxidation of indole-3-acetic acid by peroxidase

During oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase, indole-3-aldehyde and 3-hydroxymethayloxindole cease to be produced a few minutes after initiation of the reaction even though IAA is still being consumed. At the same time an increased accumulation of indole-3-methanol is observed and the ratio of oxygen to indole-3-acetic acid consumed becomes less than unity. Indole-3-niethanol can be a substrate for horseradish peroxidase provided that H₂O₂ is present. In this reaction, indole-3-aldehyde but not 3-hydroxymethyloxindole is formed. H₂O₂ is not merely an activating agent for the enzyme but also a true oxidant because it is consumed stoichiometrically (1 mol of H₂O₂ per mol of indole-3-methanol) and the reaction is independent of the presence of oxygen. Indole-3-methanol is proposed as an intermediate in the process of oxidation of indole-3-acetic acid into indole-3-al-denyde, the second step of which requires peroxide as an oxidant.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Metabolism of [14C]-indole-3-acetic acid by soybean callus and hypocotyl sections

Metabolism of indole-3-acetic acid in soybean [ Glycine max (L.) Merr.] was investigated with [1-¹⁴C]- and [2-¹⁴C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [³H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco ( Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [¹⁴C]-indole-3-methanol (IM) was three times more efficient than [2-¹⁴C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.     

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that l-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-¹⁴C] and [1-¹⁴C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than l-tryptophan.     

Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

Metabolism of Indole-3-Acetic Acid by Pericarp Discs from Immature and Mature Tomato (Lycopersicon esculentum Mill)

[1′-¹⁴C, ¹³C₆]Indole-3-acetic acid was infiltrated into immature pericarp discs from fruits of tomato (Lycopersicon esculentum Mill., cv Moneymaker). After a 24-h incubation period the discs were extracted with methanol and the partially purified extract was analyzed by reversed-phase high-performance liquid chromatography-radiocounting. Five metabolite peaks (1-5) were detected and subsequently analyzed by combined high-performance liquid chromatography-frit-fast atom bombardment-mass spectrometry. The metabolite 4 fraction was found to contain [¹³C₆]-indole-3-acetylaspartic acid, and analysis of metabolite 5 identified [¹³C₆]indole-3-acetyl-β-d-glucose. The other metabolites could not be identified, but alkaline hydrolysis studies and gel permeation chromatography indicated that metabolites 1 and 3 were both amide conjugates with a molecular weight of approximately 600. Studies with radiolabeled indole-3-acetic acid, indole-3-acetylaspartic acid, and indole-3-acetyl-β-d-glucose demonstrated that in immature pericarp indole-3-acetic acid is deactivated primarily via metabolism to indole-3-acetylaspartic acid, which is further converted to metabolites 1, 2, and 3. In mature, pink pericarp discs, indole-3-acetic acid is converted more extensively to its glucosyl conjugate. Conjugation of indole-3-acetic acid to indole-3-acetylaspartic acid appears to be dependent upon protein synthesis because it is inhibited by cycloheximide. In contrast, cycloheximide has little effect on the further conversion of indole-3-acetylaspartic acid to metabolites 1, 2, and 3.     

Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media

The characterization by capillary gas chromatography-mass spectrometry of the plant hormones indole-3-acetic acid and the gibberellins GA₁ and GA₃ from chemically-defined cultures of Acetobacter diazotrophicus and Herbaspirillum seropedicae is reported. Both bacteria are endophytic in gramineae species where they promote growth and yield. Quantification was also done by selected ion monitoring with [17,17-²H₂]-Gibberellin A₁, [17,17-²H₂]-Gibberellin A₃ and [¹³C₆]-indole-3-acetic acid as internal standards. The results presented show the importance of studying phytohormonal production when the interrelationships between plants and microorganisms are analyzed and may help explain the beneficial effects of endophytic bacteria to the host plant, as has been demonstrated previously for Azospirillum spp.     

Androgenesis in Citrus aurantifolia (Christm.) swingle

By means of gas chromatography-selected ion monitoring-mass spectrometry using an isotope-dilution assay with 4,5,6,7-tetradeutero-indole-3-acetic acid as the internal standard, indole-3-acetic acid has been estimated to be present in aseptically cultured gametophytes of wild-type Physcomitrella patens (Hedw.) B.S.G. at a level of 0.075 g g^–1 dry weight or 2.1 ng g^–1 fresh weight.Abbreviations IAA indole-3-acetic acid - d₄IAA 4,5,6,7-tetra-deutero-indole-3-acetic acid - [¹⁴C]IAA indole-3-[2-¹⁴C]-acetic acid - GC-SIM-MS gas chromatography-selected ion monitoring-mass spectrometry     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Analysis of Indole-3-Acetic Acid Metabolites from Dalbergia dolichopetala by High Performance Liquid Chromatography-Mass Spectrometry

A mixture of [2-¹⁴C₁] and [¹³C₆]indole-3-acetic acid was applied to the cotyledons of 6-day-germinated seeds of “jacarandá do cerrado” (Dalbergia dolichopetala) and after 8 hours the seeds were extracted. Analysis of the fractionated extract by reversed-phase high performance liquid chromatography-radiocounting revealed the presence of five radiolabeled metabolite peaks (I-V). After further purification, the individual peaks of radioactivity were analyzed by combined high performance liquid chromatography-steel filter-fast atom bombardment-mass spectrometry. The metabolite fraction V was found to contain [¹⁴C₁, ¹³C₆]indole-3-acetylas-partic acid and unlabeled indole-3-acetylglutamic acid. Analysis of the metabolite fraction II revealed the presence of dioxindole-3-acetylaspartic acid and putative dioxindole-3-acetylglutamic acid as well as putative benzene ring-hydroxylated derivatives of oxindole-3-acetylaspartic acid and oxindole-3-acetylglutamic acid. There was no evidence of significant incorporation of label from [2′-¹⁴C₁] or [¹³C₆]indole-3-acetic acid into any of these conjugated indoles.     

Identification of indole compounds secreted byFrankia HFPArI3 in defined culture medium

Indole compounds secreted byFrankia sp. HFPArI3 in defined culture medium were identified with gas chromatography-mass spectrometry (GC-MS). WhenFrankia was grown in the presence of¹³C(ring-labelled)-L-tryptophan,¹³C-labelled indole-3-acetic acid (IAA), indole-3-ethanol (IEtOH), indole-3-lactic acid (ILA), and indole-3-methanol (IMeOH) were identified.High performance liquid chromatography (HPLC) and GC-MS with selected ion monitoring were used to quantify levels of IAA and IEtOH inFrankia culture medium. IEtOH was present in greater abundance than IAA in every experiment. When no exogenous trp was supplied, no or only low levels of indole compounds were detected.Seedling roots ofAlnus rubra incubated in axenic conditions in the presence of indole-3-ethanol formed more lateral roots than untreated plants, indicating that IEtOH is utilized by the host plant, with physiological effects that modify patterns of root primordium initiation.     

Azido auxins : photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic Acid

Tritiated 5-azidoindole-3-acetic acid (5-N₃-[7-³H]IAA), a photoaffinity labeling agent, was used to photolabel proteins of a crude microsomal preparation from maize (Zea mays L., Bear Hybrid, WF9 × BR38) coleoptile. Approximately 50% of the bound radioactivity was solubilized in 5 molar urea containing Triton X-100, and the extract was fractionated using a variety of techniques. High performance liquid chromatography demonstrated that, although many membrane proteins incorporated tritiated label, only a few showed reduced incorporation in the presence of excess indole-3-acetic acid. By contrast, no detectable reduction in incorporation was observed in the presence of excess naphthalene-1-acetic acid. Results from isoelectric focusing gel electrophoresis indicate that the proteins that showed reduced incorporation of photolyzed 5-N₃-[7-³H]IAA in the presence of IAA fell into two main groups: one which focuses between pH 5.2 and 5.7 (pI 4.8-5.3) and another around pH 6.2 (pI 5.8). In sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins migrated as four bands with apparent molecular weights of 60, 49, 45, and 37 kilodaltons. The auxin-transport inhibitor, 2,3,5-triiodobenzoic acid, competes for the labeling by 5-N₃-[7-³H]IAA, suggesting that some of these proteins may be involved in auxin transport.     

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.