Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

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     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

Dioxindole-3-Acetic Acid Conjugates Formation from Indole-3-Acetylaspartic Acid in Vicia Seedlings

When indole-3-acetic acid (IAA) is applied to the cotyledonsof broad bean seedlings (Vicia faba L. cv Chukyo), the majormetabolites found in the roots are 3-(O-ß-glucosyl)-2-indoIone-3-acetylaspartic acid (Glc-DIA-Asp) and 3-hydroxy-2-indolone-3-acetylasparticacid (DIA-Asp). In this report, the metabolic pathway from IAAto the two dioxindole-3-acetic acid (DIA) conjugates was investigatedby using [¹⁴C]IAA, [¹⁴C]DIA, [¹⁴C]indole-3-acetylaspartic acid(IAA-Asp), and [¹⁴C]IAA-[³H]Asp. The precursor of DIA-Asp wasfound to be IAA-Asp but not DIA. Incorporation of the doublelabeled IAA-Asp into the DIA conjugates demonstrated that hydrolysisof IAA-Asp was not involved in the formation of the DIA conjugates.DIA-Asp was further metabolized to Glc-DIA-Asp in the cotyledons,while formation of Glc-DIA-Asp in the roots was very low. Glc-DIA-Aspformed in the cotyledons was transported to the roots. (Received April 21, 1986; Accepted September 10, 1986)     

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.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Identification of 3-Hydroxy-2-indolone-3-acetylaspartic Acid as a New Indole-3-acetic Acid Metabolite in Vicia Roots

A new indole-3-acetic acid (IAA) metabolite in the root of Viciafaba L. cv. Chukyo was identified as 3-hydroxy-2-indolone-3-acetylasparticacid, with the simpler name of dioxindole-3-acetylaspartic acid,by comparison with the authentic sample. Formation of dioxindole-3-aceticacid conjugates seems to be a major route of IAA metabolismin Vicia roots. (Received October 22, 1985; Accepted January 7, 1986)     

Effect of Exogenous Indole-3-Acetic Acid and Indole-3-Butyric Acid on Internal Levels of the Respective Auxins and Their Conjugation with Aspartic Acid during Adventitious Root Formation in Pea Cuttings

The influence of exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on the internal levels of these auxins was studied during the first 4 days of adventitious root formation in cuttings of Pisum sativum L. The quantitations were done by high performance liquid chromatography with spectrofluorometric detection. IBA, identified by combined gas chromatography-mass spectrometry (GC-MS), was found to naturally occur in this plant material. The root inducing ability of exogenous IBA was superior to that of IAA. The IAA level in the tissue increased considerably on the first day after application of IAA, but rapidly decreased again, returning to a level twice the control by day 3. The predominant metabolic route was conjugation with aspartic acid, as reflected by the increase in the level of indole-3-acetylaspartic acid. The IBA treatment resulted in increases in the levels of IBA, IAA, and indole-3-acetylaspartic acid. The IAA content rapidly returned to control levels, whereas the IBA level remained high throughout the experimental period. High amounts of indole-3-butyrylaspartic acid were found in the tissue after feeding with IBA. The identity of the conjugate was confirmed by ¹H-nuclear magnetic resonance and GC-MS. IBA was much more stable in solution than IAA. No IAA was detected after 48 hours, whereas 70% IBA was still recovered after this time. The relatively higher root inducing ability of IBA is ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. Adventitious root formation is discussed on the basis of these findings.     

Indole-3-butyric acid in Arabidopsis thaliana III. In vivo biosynthesis

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as one of the reaction products after incubation of sterile cultures of Arabidopsis thaliana seedlings with labeled indole-3-acetic acid (IAA). This is the first demonstration of IBA biosynthesis in a dicotyledonous plant. After 1 h of incubation most of the IBA was found in the free form, while after longer periods of incubation most of it was detected in conjugated forms. Formation of IBA conjugates was inhibited by the addition of unlabeled IBA. The biosynthesis of IBA and its conjugates was followed throughout the development of the seedlings and at different pH values. All parts of the plant (isolated roots, leaves, shoots and flowers) were able to convert IAA to IBA to the same extent.IAA was more readily transported than IBA in mature Arabidopsis plants. Feeding of labeled phenylacetic acid (PAA) and -naphthylacetic acid (NAA) to Arabidopsis seedlings resulted in a new small peak which was hydrolyzed by 7N NaOH, but the formation of compounds with longer side chains (analogous to IBA) could not be detected.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthylacetic acid - PAA phenylacetic acid     

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.     

Lateral root formation in rice (Oryza Sativa L.): differential effects of indole-3-acetic acid and indole-3-butyric acid

Comparative effects of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on lateral root (LR) formation were studied using 2-day-old seedlings of IR8 rice (Oryza sativa L.). Results showed that IBA at all concentrations (0.8–500 nmol/L) increased the number of LRs in the seminal root. However exogenous IAA, failed to increase the number of LRs. On the other hand, both IBA and IAA caused inhibition of seminal root elongation and promotion of LR elongation, but IAA can only reach to the same degree of that of IBA at a more than 20-fold concentration. Exogenous IBA had no effect on endogenous IAA content. We conclude from the results that IBA could act directly as a distinct auxin, promoting LR formation in rice, and that the signal transduction pathway for IBA is at least partially different from that for IAA.     

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.     

Identification of conjugated IAA in carrot crown gall as indole-3-acetylaspartic acid (IAAsp) by LC/MS

In carrot crown gall cells transformed with Ti plasmids, Ti-derived IAA biosynthetic genes are transcribed and translated, followed by overproduction of IAA. However, the newly synthesized IAA is immediately metabolized to IAA-amino acid conjugate, and the content of endogenous free IAA is maintained at a low level. In this study, IAA-amino acid conjugate in carrot tissues transformed with Ti plasmids was identified as indole-3-acetylaspartic acid (IAAsp) by using frit-fast atom bombardment liquid chromatography/mass spectrometry (LC/MS).     

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.     

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues

Exogenous [¹⁴C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [¹⁴C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.     

Arabidopsis Seedlings Over-Accumulated Indole-3-acetic Acid in Response to Aminooxyacetic Acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

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