Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.

     

Indole-3-methanol—a metabolite of indole-3-acetic acid in pea seedlings

Summary Degradation of indole-3-acetic acid was investigated in etiolated pea shoots; the study was limited to indolic metabolites. The products formed were fractionated by column chromatography and identified by thin-layer chromatography and chemical methods. The pathway of indole-3-acetic acid degradation involving indole-3-aldehyde was found to be more significant than stated in literature, and indole-3-methanol was established as the major indolic metabolite.The following abbreviations will be used: IAA: indole-3-acetic acid; IM: indole-3-methanol; IAld: indole-3-aldehyde; ICA: indole-3-carboxylic acid.     

Effects of sodium diethyldithiocarbamate, solvent, temperature and plant extracts on the stability of indoles

A study has been made of the effects of solvent, temperature, and the antioxidant, sodium diethyldithiocarbamate, on the breakdown of indole-3-pyruvic acid to indole-3-acetic acid (IAA). In addition, the degradation of tryptophan, tryptamine, indole-3-pyruvic acid, indole-3-acetaldehyde and indole-3-ethanol to IAA during the purification and analysis of extracts from Pinus sylvestris L. needles, in the presence and absence of sodium diethyldithiocarbamate, has been investigated. The data obtained indicate that if the antioxidant is supplied throughout the analytical sequence there is a marked reduction in the spontaneous formation of IAA from other indolic compounds and, by inference, the stability of indoles in general is enhanced.     

Metabolism of Auxin in Tomato Fruit Tissue: Formation of High Molecular Weight Conjugates of Oxindole-3-Acetic Acid via the Oxidation of Indole-3-Acetylaspartic Acid

High performance liquid chromatography of extracts of tomato (Lycopersicon esculentum Mill.) incubated with a relatively low concentration (4 μm) of [1-¹⁴C]indole-3-acetic acid (IAA) revealed the presence of two major polar metabolites. Hydrolysis of the two metabolites with 7 n NaOH yielded the same compound, which had a retention time similar to that of ring-expanded oxindole-3-acetic acid (OxIAA) on high performance liquid chromatography. The identity of the indolic moiety of these conjugates as OxIAA was further confirmed by gas chromatography-mass spectrometry. Chromatography of the two OxIAA conjugates on a calibrated Bio-Gel P-2 column indicated that their molecular weights are about 1200 and 1000. Aspartic acid and glutamic acid were the major amino acids detected in acid hydrolysates of the two conjugates. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of indole-3-acetylaspartic acid (IAAsp) with a concomitant decrease in the formation of the two OxIAA conjugates. Feeding experiments with labeled IAAsp and OxIAA showed that IAAsp and not OxIAA is the precursor of these conjugates. The data obtained indicate that exogenous IAA is converted in tomato pericarp tissue to high molecular weight conjugates, presumably peptides, of OxIAA via the oxidation of IAAsp. The oxidation of IAAsp seems to be a rate-limiting step in the formation of these conjugates from exogenous IAA.     

Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla.

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.     

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.     

Metabolism of indole-3-acetic acid by orange (Citrus sinensis) flavedo tissue during fruit development

[5-3H, 1'-14C, 13C6, 12C] Indole-3-acetic acid (IAA), was applied to the flavedo (epicarp) of intact orange fruits at different stages of development. After incubation in the dark, at 25 degrees C, the tissue was extracted with MeOH and the partially purified extracts were analyzed by reversed phase HPLC-RC. Six major metabolite peaks were detected and subsequently analyzed by combined HPLC-frit-FAB MS. The metabolite peak 6 contained oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp) and also indole-3-acetyl-N-glutamic acid (IAGlu). The nature of metabolite 5 remains unknown. Metabolites 3 and 4 were diastereomers of oxindole-3-acetyl-N-aspartic acid (OxIAAsp). Metabolite 2 was identified as dioxindole-3-acetic acid and metabolite 1 as a DiOx-IAA linked in position three to a hexose, which is suggested to be 3-(-O-beta-glucosyl) dioxindole-3-acetic acid (DiOxIAGlc). Identification work as well as feeding experiments with the [5-3H]IAA labeled metabolites suggest that IAA is metabolized in flavedo tissue mainly through two pathways, namely IAA-OxIAA-DiOxIAA-DiOxIAGlc and IAA-IAAsp-OxIAAsp. The flavedo of citrus fruit has a high capacity for IAA catabolism until the beginning of fruit senescence, with the major route having DiOxIAGlc as end product. This capacity is operative even at high IAA concentrations and is accelerated by pretreatment with the synthetic auxins 2,4-D, NAA and the gibberellin GA3.     

Isolation of Indole-3-Acetyl Amino Acids using Polyvinylpolypyrrolidone Chromatography

Amino acid conjugates of IAA can be chromatographed on polyvinylpolypyrrolidone (PVPP) using either acidic methanol or aqueous buffers as eluents. In the aqueous system, elution of the compounds is pH-dependent, and the pattern obtained suggests that hydrophobic interactions contribute substantially to the chromatographic behavior of IAA peptides on PVPP. Purification of soybean seed extracts by PVPP chromatography produced fractions containing indole-3-acetylaspartate and indole-3-acetylglutamate, based on chromatographic and mass spectral analysis, as well as three other indolic compounds, tentatively identified as N-acyl tryptophan derivatives. PVPP chromatography provides an effective preliminary purification of IAA peptides from plant extracts prior to their separation by other techniques such as high performance liquid chromatography.     

A Novel Metabolic Pathway for Indole-3-Acetic Acid in Apical Shoots of Populus tremula (L.) x Populus tremuloides (Michx.)

Metabolism of indole-3-acetic acid (IAA) in apical shoots of Populus tremula (L.) x Populus tremuloides (Michx.) was investigated by feeding a mixture of [12C]IAA, [13C6]IAA, and [1[prime]-14C]IAA through the base of the excised stem. HPLC of methanolic plant extracts revealed eight major radiolabeled metabolites after a 24-h incubation period. Comparison between feeds with [5-3H]IAA and [1[prime]-14C]IAA showed that all detectable metabolites were nondecarboxylative products. The purified radiolabeled HPLC fractions were screened by frit-fast atom bombardment liquid chromatography-mass spectrometry for compounds with characteristic fragment pairs originating from the application with 12C and 13C isotopes. Samples of interest were further characterized by gas chromatography-mass spectrometry. Using this procedure, oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp), oxindole-3-acetyl-N-aspartic acid (OxIAAsp), and ring-hydroxylated oxindole-3-acetic acid were all identified as IAA metabolites. Furthermore, a novel metabolic pathway from IAA via IAAsp and OxIAAsp to OxIAA was established on the basis of refeeding experiments with the different IAA metabolites.     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

Quantification of free plus conjugated indoleacetic acid in arabidopsis requires correction for the nonenzymatic conversion of indolic nitriles.

The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and indentification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.     

Sur la dégradation de l'acide indolyl-3-acétique par Nectria galligena Bres. var. major Wr.

Summary Nectria galligena is grown in synthetic medium. Experiments are carried out with suspensions of washed mycelium, incubated and stirred with indole-3-acetic acid (IAA) or IAA 2-¹⁴C. Auxin degradation is quicker with either acid pH or young mycelium. Two indolic compounds are identified in the course of this metabolism: indole-3-aldehyde (IAld) and indole-3-carboxylic acid (ICA).A correlation is supposed to exist between the increase of auxin contained in cultures of Nectria and IAA catabolism as it lessens with age and alcalin pH.

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Dans le texte les abréviations suivantes seront employées IAA Acide Indolyl-3-acétique - IAld Indolyl-3-aldéhyde - ICA Acide Indolyl-3-carboxylique - DPH 2,4-dinitrophénylhydrazine - DMCA Paradiméthylaminocinnamaldéhyde     

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.     

L'acide indolyl-3-lactique et son métabolisme chez Rhizobium

Summary Among the indole compounds formed when tryptophan 2-¹⁴C is metabolized by Rhizobium, indole-3-lactic acid (ILA) is specially studied. In the course of experiments carried out in the culture medium of growing Rhizobium and in suspensions of washed bacterial cells the amount of ILA formed is compared with that of indole-3-acetic acid (IAA) occurring simulataneously. The formation of ILA and that of IAA directly depend on a transamination reaction. A large quantity of ILA is present in suspensions of washed bacterial cells.When ILA alone, as precursor, is incubated with Rhizobium, several products are identified: IAA, indole-3-acetaldehyde and tryptophol. Tryptophan is also detected in the aqueous fraction and is labelled when ILA 2-¹⁴C is used. The pathway of this metabolism are discussed and a general scheme is suggested.     

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     

The role of the epidermis in auxin-induced and fusicoccin-induced elongation of Pisum sativum stem segments

The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 10^-8 M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

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.