Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

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     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using ¹³C₆-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).     

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

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.     

Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plants

A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-¹⁴C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production. The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.     

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.     

Purification of indole-3-acetic acid in plant extracts by immunoaffinity chromatography

Polyclonal rabbit antiserum, raised against IAA coupled to bovine serum albumen via the indole nitrogen, was purified on a Protein A column. The immunoglobulin fraction was covalently bound to glutardialdehyde-activated silicate support and used as an immunoaffinity chromatography matrix to purify IAA in extracts from the cambial zone and shoots of Pinus sylvestris. Samples were then analysed by reverse phase HPLC with fluorescence detection. The accuracy of quantitative estimates of IAA, based on isotope dilution analyses, were verified by means of a successive approximation. The presence of IAA in the cambial tissue was further confirmed by GC/MS.     

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.     

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.Abbreviations 4Cl-IAA 4-chloro-indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA Indole-3-acetic acid - IAA-C5-BSA, IAA-C1-BSA, IAA-NI-BSA hapten-carrier-conjugates with IAA linked to bovine serum albumin through the C5-position, the carboxyl group, and the indole nitrogen, respectively - IAA-Me the methylester of IAA This study was supported by the Danish Research Council (SJVF 13-4148 and 13-4547 to P.U.) and by The Research Center for Plant Biotechnology.     

Gas chromatography-mass spectrometry evidence for several endogenous auxins in pea seedling organs

Qualitative analysis by gas chromatography-mass spectrometry (GC-MS) of the auxins present in the root, cotyledons and epicotyl of 3-dold etiolated pea (Pisum sativum L., cv. Alaska) seedlings has shown that all three organs contain phenylacetic acid (PAA), 3-indoleacetic acid (IAA) and 4-chloro-3-indoleacetic acid (4Cl-IAA). In addition, 3-indolepropionic acid (IPA) was present in the root and 3-indolebutyric acid (IBA) was detected in both root and epicotyl. Phenylacetic acid, IAA and IPA were measured quantitatively in the three organs by GC-MS-single ion monitoring, using deuterated internal standards. Levels of IAA were found to range from 13 to 115 pmol g^-1 FW, while amounts of PAA were considerably higher (347–451 pmol g^-1 FW) and the level of IPA was quite low (5 pmol g^-1 FW). On a molar basis the PAA:IAA ratio in the whole seedling was approx. 15:1.Abbreviations IAA 3-indoleacetic acid - 4Cl-IAA 4-chloro-3-indoleacetic acid - IBA 3-indolebutyric acid - IPA 3-indolepropionic acid - PAA phenylacetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFB pentafluorobenzyl ester - PFBBr pentafluorobenzyl bromide - SIM single-ion monitoring - TMSI trimethylsilyl ester     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

Identification of Indole-3-Acetic Acid in the Basidiomycete Schizophyllum commune

Indole-3-acetic acid (IAA) was detected in the ether extracts of culture filtrates of indigotin-producing strains of the basidiomycete Schizophyllum commune. Several solvents, known to give distinctly different R_F values for IAA, and 3 location reagents gave identical results with synthetic IAA and IAA found in the extract. Confirmation was obtained by the Avena straight growth test, split pea test, and ultraviolet absorption spectrum.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

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.     

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.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.