The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts

Different types of plant material, including both dry and swollen maize kernels, swollen bean seeds, bean seedlings and dry rose seeds, were extracted by different methods and the yield of IAA was determined with the indolo-α-pyrone method. Extraction of dry maize kernels during short time experiments, varying from 3 to 24 h, gave the highest IAA yield when methanol was the extractant and a significant lower yield when diethyl ether or dichloromethane were used. The duration of the extraction period increased the yield with all the extractants. Progressive extractions for several days or weeks had little effect on the yield when 100% acetone was used in contrast to methanol and ether as extractants, which increased the yield during prolonged extraction. Extractions of tissue treated to 100°C for 1 h contradicted the hypothesis that IAA is enzymatically liberated during ether extraction. Water in the extractant solvents increased the yields. This was most pronounced when aqueous acetone was used instead of 100% acetone. Increased extraction temperature augmented the IAA yields. The yield of IAA from other types of tissue extracted with methanol for periods of 3 or 24 h was, however, independent of the duration of the extraction time. This indicates that some tissues contain less not easily extractable IAA than dry maize kernels. The terms “free” and “bound” IAA are discussed; they should be replaced by “easily extractable” and “not easily extractable” IAA. The results also show that IPyA in vitro can partly be converted to IAA during extraction and fractionation.     

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     

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.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Indole-3-acetic acid in Douglas fir seedlings: A reappraisal

The use of ring-labelled, pentadeutero IAA as an internal standard in selected ion monitoring analysis of Douglas fir seedlings revealed an estimate of IAA which was nearly an order of magnitude smaller than that reported earlier.     

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.     

Indole-3-acetic acid in microbial and microorganism-plant signaling

Diverse bacterial species possess the ability to produce the auxin phytohormone indole-3-acetic acid (IAA). Different biosynthesis pathways have been identified and redundancy for IAA biosynthesis is widespread among plant-associated bacteria. Interactions between IAA-producing bacteria and plants lead to diverse outcomes on the plant side, varying from pathogenesis to phyto-stimulation. Reviewing the role of bacterial IAA in different microorganism-plant interactions highlights the fact that bacteria use this phytohormone to interact with plants as part of their colonization strategy, including phyto-stimulation and circumvention of basal plant defense mechanisms. Moreover, several recent reports indicate that IAA can also be a signaling molecule in bacteria and therefore can have a direct effect on bacterial physiology. This review discusses past and recent data, and emerging views on IAA, a well-known phytohormone, as a microbial metabolic and signaling molecule.     

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     

Catabolism of indole-3-acetic acid in citrus leaves: identification and characterization of indole-3-aldehyde oxidase

A new enzyme, named indole-3-aldehyde oxidase (IAldO), was identified in citrus ( Citrus sinensis L. Osbeck cv. Shamouti) leaves. The enzyme was partially purified by (NH₄)₂SO₄ fractionation. Sephadex G-200 gel filtration and DEAE-cellulose ion exchange chromatography. IAldO catalyzes the oxidation of indole-3-aldehyde (IAld) to indole-3-carboxylic acid (ICA) with the production of H₂O₂. The enzyme is highly specific for IAld. The apparent K_M of the enzyme for IAld is 19 μ M . The optimum oxidation of IAld occurs at pH 7. 5. The molecular mass of the enzyme, as determined by Sepharose-6B gel filtration, is about 200 kDa. Based on inhibitor studies, it is concluded that IAldO is not a flavin-linked oxidase and there is no requirement for free sulfhydryl groups or divalent cations for maximum activity. The enzyme is strongly inhibited by benzaldehyde. Ethylene pretreatment, wounding and aging of leaf tissues did not affect enzyme activity, suggesting that the enzyme is constitutive in citrus tissues.     

New phenolic inhibitors of the peroxidse-catalysed oxidation of indole-3-acetic acid

Previously we reported two metabolites of the insecticide carbofuran as persistent inhibitors of the peroxidase-catalysed oxidtion ofindole-3-acetic acid. In searching for more active inhibitors of this type, we have found that 5-hydroxy-2,2-dimethylchromene (β-tubanol), 2′,6′-dihydroxycetophenone oxime, 5-hydroxy-2,2-dimethylchroman, 2′,6′-dihydroxyacetophenone and 2,6-dihydroxybenzoic acid methyl ester were more active than the carbofuran metabolite 7-hydroxy-2,2-dimethyl-3-oxo-2,3-dihydrobenzofuran. Resorcinol, 5-hydroxy-2,2-dimethylchroman-4-one, 3-hydroxy-5-methoxy-2,2-dimethylchroman-4-one and 5-hydroxy-2-methylchrom-4-one were also inhibitory but with less activity. The new inhibitors differed from the well-known phenolic inhibitors such as caffeic acid in inhibition kinetics as demonstrated by the rate of disappearance of indole-3-acetic acid, the rate of formation of the oxidation products, and the transient spectral change in the enzyme.     

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.     

Indole-3-acetic acid: A widespread physiological code in interactions of fungi with other organisms

Plants as well as microorganisms, including bacteria and fungi, produce indole-3-acetic acid (IAA). IAA is the most common plant hormone of the auxin class and it regulates various aspects of plant growth and development. Thus, research is underway globally to exploit the potential for developing IAA-producing fungi for promoting plant growth and protection for sustainable agriculture. Phylogenetic evidence suggests that IAA biosynthesis evolved independently in bacteria, microalgae, fungi, and plants. Present studies show that IAA regulates the physiological response and gene expression in these microorganisms. The convergent evolution of IAA production leads to the hypothesis that natural selection might have favored IAA as a widespread physiological code in these microorganisms and their interactions. We summarize recent studies of IAA biosynthetic pathways and discuss the role of IAA in fungal ecology.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

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.     

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.