In vitro binding affinities of 4-chloro-, 2-methyl-, 4-methyl-, and 4-ethylindoleacetic acid to auxin-binding protein 1 (ABP1) correlate with their growth-stimulating activities

4-Chlorindole-3-acetic acid (4-CI-IAA), an endogenous auxin in certain plant species of Fabaceae, has a higher efficiency in stimulating cell elongation of grass coleoptiles compared with indole-3-acetic acid (IAA), particularly at low concentrations. However, some investigations reported a 1,000-fold discrepancy between growth stimulation and binding affinity of 4-CI-IAA to auxin-binding protein 1 (ABP1) from maize. Here we report binding data of 4-CI-IAA and three alkylated IAA derivatives using purified ABP1 in equilibrium dialysis. There is a clear correlation between the growth-promoting effects and the binding affinity to ABP1 of the different IAA analogues measured by competition of [³H]naphthalene-1-acetic acid binding. Our data are consistent with the hypothesis that ABP1 mediates auxin-induced cell elongation.Abbreviations ABP1 auxin-binding protein 1 - 4-CI-IAA 4-chloroindole-3-acetic acid - NAA naphthalene-1-acetic acid - ER endoplasmic reticulum - IAA indole-3-acetic acid - 2-Me-IAA 2-methylindole-3-acetic acid - 4-Me-IAA 4-methylindole-3-acetic acid - 4-Et-IAA 4-ethylindole-3-acetic acid - MES 4-morpholineethanesulfonic acid - PAA phenylacetic acid     

Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition of Exogenous IAA in Arabidopsis thaliana

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D-glucopyranose, [2′,2′-²H₂]-2-oxoindole-3-acetic acid, and 1-O-([2′,2′-²H₂]-2-oxoindole-3-acetyl)-β-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

Summary The kinetics of MT reorientation in primary roots ofZea mays cv. Merit, were examined 15,30,45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4–5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10^–9 to 10^–6M IAA. IAA treatment at 10^–8M or less showed no major or consistent changes but 10^–7 M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10^–5M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether) N,N,NN-tetraacetic acid - MTs cortical microtubules - QC quiescent center - MES/TRIS 2-(N-morpholino)ethanesulfonic acid/tris(hydroxymethyl)aminomethane - IAA indole-3-acetic acid - PBS phosphate buffered saline - PHEMD [60 mM Pipes (piperazine-diethanesulfonic acid), 25 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 10 mM EGTA, 2mM MgCl₂ pH7.0 adjusted with NaOH] containing 5% dimethyl sulfoxide     

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     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid     

The Effects of Growth Regulators on Flowering of Chenopodium murale Plants in vitro

In vitro culture of Chenopodium murale L. (ecotype 197) green and herbicide SAN 9789 - treated "white" plants was established and the effects of benzylaminopurine (BAP), indole-3-acetic acid (IAA) and gibberellic acid (GA₃) on growth and flowering were tested. Green plants did not flower on glucose free media, while 17 % of plants flowered on 5 % glucose-containing medium. SAN 9789 (10^–5 M) inhibited growth and flowering. BAP and IAA (0.1 – 5 mg dm^–3) also inhibited growth and flowering of green and "white" plants. GA₃ (10 mg dm^–3) stimulated leaf development in green plants, but had no significant effect on "white" plants, and stimulated flowering of green (41 %) and "white" (33 %) plants.     

A mutation affecting the synthesis of 4-chloroindole-3-acetic acid

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana,¹ although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further.² Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.     

(+)-5-Hydroxy-dioxindole-3-acetic acid,a synergist from rice bran of auxin-induced ethylene production in plant tissue

(+)-5-Hydroxy-dioxindole-3-acetic acid (1) was isolated from rice bran as a substance synergistic with auxin in the auxin induced ethylene production by etiolated mungbean hypocotyl segments. 5-Hydroxy-oxindole-3-acetic acid (4) and IAA were also obtained. The importance of a hydroxyl group in the 5-position in the two compounds was suggested since synthesized (±)-dioxindole-3-acetic acid (6) was inactive.     

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.     

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.     

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [³H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5–5 M IAA is included, the pulse achieves its maximum velocity of about 2 cm h^-1. At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [³H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A^-) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [³H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A^-. The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A^- component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.Abbreviations A^- auxin anion - HA undissociated auxin - IAA indole-3-acetic acid - NPA N-1-napthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

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.     

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     

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.).     

Surface-enhanced Raman spectroscopy combined with atomic force microscopy for ultrasensitive detection of thrombin

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA–Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC–MS/MS-based assay yields the following parameters: V/E_t^IAA = 20.3 min⁻¹, K_m^IAA = 123 μM, V/E_t^ATP = 14.1 min⁻¹, K_m^ATP = 50 μM, V/E_t^Asp = 28.8 min⁻¹, K_m^Asp = 1580 μM. This is the first assignment of kinetic values for any IAA–amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC–MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.     

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 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     

Quantitative predictions for the chemiosmotic uptake of auxin

1. The predictions of a general kinetic model for the chemiosmotic uptake of auxin and other weak acids are compared with experimental results for the auxin indoleacetic acid. The proposed mechanism involves diffusional flux of undissociated acid, a saturable, voltage-sensitive flux of anion (A^-), and a carrier-mediated symport of H⁺ and A^-, all operating in parallel. During much of uptake, the electrochemical gradients are such that the net symport and the net anion flux are in opposition: the symport contributes more to influx; the anion path, to efflux. The voltage-sensitive flux of A^- therefore constitutes a leak. 2. The presence of a symport, whose carrier can distribute across the membrane in response to the internal and external concentrations of auxin, can speed the rate of uptake, but does not by itself alter the accumulation of auxin at equilibrium. 3. The accumulation ratio at equilibrium is less at low concentrations of auxin than at higher concentrations, indicating the presence of a saturable anion path. The concentration dependence of the transition depends on several factors, and is not a reliable indicator of the A^--carrier binding constant. 4. Observed uptake near neutral pH appears larger than is consistent with a voltage-sensitive anion flux being the only carrier-mediated path across the membrane. This observation provides indirect evidence for the presence of an auxin-proton symport in addition to a saturable A^- carrier. 5. The change in kinetics of uptake of [³H]indole-3-acetic acid (IAA), observed as the total concentration of IAA is raised from 0.1 to 100 M, is consistent with either (i) a symport that saturates at low concentrations, or (ii) activation of an A^- efflux by intermediate concentrations of auxin. 6. The data on the concentration dependence of uptake of auxin are not consistent with a multi-proton symport.Abbreviations A^- auxin anion - HA weak acid, particularly IAA - HXA carrier in electroneutral complex with a proton and the auxin anion - H₂XA carrier in electroneutral complex with two protons and the auxin anion - IAA indole-3-acetic acid - X auxin carrier - XA carrier-auxin anion complex