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.     

Differentiation between indoleacetic Acid and the citrus auxin by column chromatography

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.     

Auxin-conjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease

Auxin conjugates play a role in the regulation of free indole-3-acetic acid (IAA) content in plants. Not much is known about the enzymes involved in either conjugate synthesis or hydrolysis. In this study we have isolated and characterized an auxin conjugate hydrolase from Chinese cabbage seedlings and investigated it during the development of both the Chinese cabbage plants and the clubroot disease. The hydrolase isolated from light- and dark-grown seedlings accepted the amide conjugates indole-3-acetic acid-alanine (IAAla), IAA-phenylalanine (IAPhe), but not IAA-aspartate (IAAsp) as substrates. We also found a substantial amount of hydrolysis of an ester conjugate (IAA-glucose, IAGlu) in our enzyme preparation. The tentative reaction product IAA was identified by HPLC and subsequent GC-MS analysis. The pH optima for the different substrates were not identical, suggesting several hydrolase isoforms. After gel filtration chromatography we found at least two peaks containing different hydrolase isoforms. The isoform, which converted IAGlu to IAA, exhibited a molecular mass of ca 63 kDa, and an isoform of ca 21 kDa converted IAAla and IAPhe. The increased free IAA content in clubroot-diseased roots of Brassicaceae can be due to either de novo synthesis or release of IAA from conjugates. To answer this question free, ester- and amide-bound IAA was measured in 24- and 30-day-old leaves and roots of healthy and Plasmodiophora brassicae-infected Chinese cabbage, and the hydrolase activity with different substrates measured in the same tissues. The amide conjugates were dramatically enhanced in infected roots, whereas free IAA was only slightly enhanced compared to the control tissue. Hydrolase activity was also enhanced in clubbed roots, but the substrate specificity differed from that found in the seedlings. Especially, IAAsp hydrolysis was induced after inoculation with P. brassicae. We conclude that different auxin conjugates can be hydrolyzed at different developmental stages or under stress.     

Identification of phenylacetic acid as a natural auxin in the shoots of higher plants

Evidence indicating the natural occurrence of the auxin substance, phenylacetic acid (PAA), in a range of crop plants has been obtained from paper chromatography followed by bioassay and from HPLC and GLC analysis of acidic ether extracts from vegetative shoots of these plants. Confirmatory evidence for the presence of PAA in tobacco shoots has been obtained from GC-MS analysis. Quantitative estimation of the relative amounts of the two auxins, IAA and PAA, in the different shoot extracts was achieved by paper chromatography followed by gas chromatography. The amount of PAA in all six plants was found to be several times greater than that of IAA and calculation of average internal concentrations revealed that PAA is present in vegetative shoots at physiologically active concentrations. Present knowledge of the growth-regulating activity of this new natural auxin is discussed.     

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.     

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.     

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.     

Auxin in scapes,flower buds,flowers, and fruits of daffodil (Narcissus pseudonarcissus L.)

Summary Diffusates from flower buds, flower fruits, and scape segments, and extracts of flower stalks of Narcissus pseudonarcissus contain an auxin active in the Avena geo-curvature test. The auxin behaved like indole-3-acetic acid (IAA) in thin-layer chromatography (TLC) with neutral and basic solvents on different adsorbents. After TLC, the auxin of the extracts showed chromogenic reactions identical with those of IAA; in gas-liquid chromatography on two different columns, the purified substance, after methylation, appeared at the retention time of IAA methyl ester. The auxin content of the extracts has been estimated to be equivalent to ca. 10 g IAA kg^–1 fresh weight. Diffusates, collected at the basal end of excised flowering apices and of scape segments at different developmental stages, showed highest auxin activity when collected from old buds and young flowers, and from the basal, rapidly elongating scape regions. The diffusible auxin obtained from scape segments was very likely produced by the segments themselves. Thus, the shoot of Narcissus appears to possess two different sites of auxin production, namely, the apical region represented by the flower bud, the flower or the fruit, and the scape.Abbreviations IAA indole-3-acetic acid - IAA-OMe indole-3-acetic-acid methyl ester - TLC thin-layer chromatography - GLC gas-liquid chromatography     

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.     

Tryptophan-dependent biosynthesis of auxins in soil

The presence of auxins in soil may have an ecological impact affecting plant growth and development. A rapid and simple colorimetric method was used to assess California soils for their potential to produce auxins upon the addition of L-tryptophan (L-TRP). The auxin content measured by colorimetry was expressed as indole-3-acetic acid (IAA)-equivalents. A substrate (L-TRP) concentration of 5.3 g kg^-1, glucose concentration of 6.7 g kg^-1, no nitrogen, pH 7.0, 40°C, shaking (aeration) and 48 h incubation time were selected as standardized conditions to assay for auxin biosynthesis in soil. IAA was confirmed as a major microbial metabolite derived from L-TRP in soil by use of high performance liquid chromatography (HPLC). Under standardized conditions, L-TRP-derived auxins in 19 soils varied greatly ranging from 18.2 to 303.2 mg IAA equivalents (auxins) kg^-1 soil. This study suggests that the phenotypic character of the soil microbiota has more of an influence on auxin production than the soil physicochemical properties (e.g., pH, organic C content, CEC, etc.).     

Production of indole-3-acetic acid by several wild-type strains of Ustilago maydis

Indole-3-acetic acid (IAA) was identified and quantitated in spent media from cultures of ten Ustilago maydis strains. IAA was identified by thin-layer chromatography, high performance liquid chromatography (HPLC) and u.v. spectroscopy, and was quantitated by HPLC. All strains produced IAA in a tryptophan (Trp)-supplemented minimal medium at levels of 0.1 to 4.0 g IAA/ml of spent medium as assessed by HPLC. The highest levels of IAA were found in strains I2 and P2. The latter was also capable of producing IAA without addition of Trp to the medium.     

Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux

The phytohormone auxin is involved in the regulation of a variety of developmental processes. In this report, we describe how the processes of lateral root and root hair formations and root gravity response in rice are controlled by auxin. We use a rice mutant aem1 (auxin efflux mutant) because the mutant is defective in these characters. The aem1 line was originally isolated as a short lateral root mutant, but we found that the mutant has a defect in auxin efflux in roots. The acropetal and basipetal indole-3-acetic acid (IAA) transports were reduced in aem1 roots compared to wild type (WT). Furthermore, gravitropic bending as well as efflux of radioactive IAA was impaired in the mutant roots. We also propose a unique distribution of endogenous IAA in aem1 roots. An immunoassay revealed a 4-fold-endogenous IAA content in the aem1 roots compared to WT, and the application of IAA to the shoot of WT seedlings mimicked the short lateral root phenotype of aem1, suggesting that the high content of IAA in aem1 roots impaired the elongation of lateral roots. However, the high level of IAA in aem1 roots contradicts the auxin requirement for root hair formation in the epidermis of mutant roots. Since the reduced development in root hairs of aem1 roots was rescued by exogenous auxin, the auxin level in the epidermis is likely to be sub-optimum in aem1 roots. This discrepancy can be solved by the ideas that IAA level is higher in the stele and lower in the epidermis of aem1 roots compared to WT and that the unique distribution of IAA in aem1 roots is induced by the defect in auxin efflux. All these results suggest that AEM1 may encode a component of auxin efflux carrier in rice and that the defects in lateral roots, root hair formation and root gravity response in aem1 mutant are due to the altered auxin efflux in roots.     

Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μ M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants ( gluS ) deficient in chloroplastic glutamate synthase.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Estimates of free and bound indole-3-acetic acid and zeatin levels in relation to regulation of apical dominance and tiller release in oat shoots

Oat stem segments containing quiescent lateral (tiller) buds during times of strong apical dominance, and growing buds released from this inhibition, were collected for analysis of native auxin and cytokinins. Free IAA and IAA conjugates were determined by a¹⁴C-IAA and¹⁴C-IBA double isotope dilution assay. Free zeatin (Z), zeatin riboside (Z-r), and their glucoside conjugates were purified from butanol-soluble fractions by means of a cellulose phosphate exchanger and thin-layer chromatography. Hormones were analyzed by gas chromatography and mass spectrometry (GC-MS). Results of these analyses indicate that changes in free and bound IAA within the stem do not correlate well with the release of tiller buds (as brought about by decapitation, gravistimulation, or the emergence of the inflorescence). However, increases in Z-r levels are well correlated with tiller release. The glucoside conjugate of Z-r may act as a storage form of cytokinin in quiescent tiller buds. In light of these results, we find that the auxin-cytokinin ratio in oat stem segments is shifted during tiller     

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard

Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with ¹³C₆[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned.