Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.     

Photo-regulation of the ratio of ester to free indole-3-acetic acid

A light exposure, sufficient to cause a 30% reduction in growth rate of seedlings of Zea mays, causes a decrease of 40% in the concentration of free indole-3-acetic acid in the seedling and an increase in the content of esterified indole-3-acetic acid. We conclude that one mechanism for regulation of plant growth is alteration of the ratio of free to conjugated hormone by environmental stimuli.     

Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid.

From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-beta-D-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-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.     

A family of auxin-conjugate hydrolases that contributes to free indole-3-acetic acid levels during Arabidopsis germination

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-beta-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.     

Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.     

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.     

Transport of indole-3-butyric acid and indole-3-acetic acid in Arabidopsis hypocotyls using stable isotope labeling

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.     

Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana

Three metabolites of indole-3-acetic acid (IAA), N-(6-hydroxyindol-3-ylacetyl)-phenylalanine (6-OH-IAA-Phe), N-(6-hydroxyindol-3-ylacetyl)-valine (6-OH-IAA-Val), and 1-O-(2-oxoindol-3-ylacetyl)-beta-d-glucopyranose (OxIAA-Glc), were found by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based search for oxidative IAA metabolites during the vegetative growth of Arabidopsis. Their structures were confirmed by making a comparison of chromatographic characteristics and mass spectra between naturally occurring compounds and synthetic standards. An incorporation study using deuterium-labeled compounds showed that 6-OH-IAA-Phe and 6-OH-IAA-Val were biosynthesized from IAA-Phe and IAA-Val, respectively, which strongly suggested the formation of these amino acid conjugates of IAA in plants. Both 6-OH-IAA-Phe and 6-OH-IAA-Val were inactive as auxins, as indicated by no significant root growth inhibition in Arabidopsis. Quantitative analysis demonstrated that OxIAA-Glc was present in the largest amount among the metabolites of IAA in Arabidopsis, suggesting that the conversion into OxIAA-Glc represents the main metabolic process regarding IAA in Arabidopsis.     

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Interaction between aspterric acid and indole-3-acetic acid on reproductive growth in Arabidopsis thaliana

Application of indole-3-acetic acid (IAA) with a pollen growth inhibitor, aspterric acid (AA), results in the recovery of normal pollen development. In contrast, application of gibberellin (GA3) with AA do not induce normal pollen growth. In addition, application of different concentrations of IAA with AA shortens the period of growth from bolting to first flowering as compared to that treated with AA alone. Furthermore, stem length and number of flower bud treated with IAA and AA were similar to those of control. These results suggest, that IAA may play an important role in reproductive growth of A. thaliana.     

Uptake and metabolism of indole-3-butyric acid and indole-3-acetic acid by Petunia cell suspension culture

The uptake and metabolism of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were studied in suspension cell cultures of Petunia hybrida. The initial uptake of ³H-IBA was much higher than that of ³H-IAA, and after 10 min of incubation with labeled IBA and IAA, 4.6 pM vs 0.35 (39% vs 12% of total applied radioactivity) respectively, were found in the cell extracts. The uptake of IBA reached a plateau of 6.0 pM (62%) after 2 h while that of IAA increased continuously up to 1.5 pM (46%) after 24 h. Following the addition of 40 µM of unlabeled auxin more IBA was taken in initially than IAA (39% vs 12%), but the level almost equalized after 24 h of incubation when IBA uptake reached 890 nM (55%) and IAA 840 nM (46%).IBA was metabolized very rapidly by Petunia cell suspension to new compounds. HPLC of the cell extracts demonstrated a new metabolite after only 2 min of incubation, and after 30 min 60% of the radioactivity was in the new metabolite vs 10% in the IBA. The new compound was resolved by autofluorography to two metabolites but after 24 h only one metabolite was present. The IBA metabolites were identified tentatively as IBA aspartic acid (IBAasp) and IBA glucose (IBAglu). In the medium IBA disappeared at a fast rate and after 24h most of the radioactivity was present in the new metabolite, probably IBAasp. IAA was also converted rapidly to two new metabolites and both were still present after 24 h. No attempt was made to identify the metabolites of IAA. IAA metabolism proceeded at a slower rate, and autofluorography showed that while free IBA disappeared after 0.5 h, free IAA was still present after 1 h of incubation. We postulate that Petunia cells conjugate IBA rapidly to IBAglu which in turn is converted to form IBAasp which probably acts as a slow release hormone. Only intact cells were able to metabolize IBA and the reaction was affected by low temperature and anaerobic conditions. The fast rate of IBA uptake, the need for whole cells for the metabolism to proceed, and the fast change of IBA to a new metabolite in the medium, all suggest that both uptake and metabolism of IBA in Petunia cells occur on the cell surface.     

Possibilities to reduce adverse effects of salinity by indole-3-acetic acid

Salinity caused a consistent reduction in the growth of cowpea plants and water content in their leaves. The total as well as the pigment fractions, except carotenoids, exhibited lower values than those of control plants at almost all salinity levels. With the rise of salinization, the total nitrogen and potassium contents in the leaves were decreased but the sodium content was increased and phosphorus content was not significantly affected as compared with the controls. The application of IAA to salt-treated plants increased the water content in the leaves but it had no effect on the number of leaves and the stem length. The pigment contents in the leaves were either promoted or inhibited with the application of IAA, depending upon the level of salinization. The application of IAA to plants irrigated with water of the highest salinity level was effective in increasing the potassium content in the leaves as compared with the control, but it had no effect on the other mineral elements.     

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     

Metabolism of indole-3-acetic acid in rice: identification and characterization of N-beta-D-glucopyranosyl indole-3-acetic acid and its conjugates

A search was made for conjugates of indole-3-acetic acid (IAA) in rice (Oryza sativa) using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in order to elucidate unknown metabolic pathways for IAA. N-beta-d-Glucopyranosyl indole-3-acetic acid (IAA-N-Glc) was found in an alkaline hydrolysate of rice extract. A quantitative analysis of 3-week-old rice demonstrated that the total amount of IAA-N-Glc was equal to that of IAA. A LC-ESI-MS/MS-based analysis established that the major part of IAA-N-Glc was present as bound forms with aspartate and glutamate. Their levels were in good agreement with the total amount of IAA-N-Glc during the vegetative growth of rice. Further detailed analysis showed that both conjugates highly accumulated in the root. The free form of IAA-N-Glc accounted for 60% of the total in seeds but could not be detected in the vegetative tissue. An incorporation study using deuterium-labeled compounds showed that the amino acid conjugates of IAA-N-Glc were biosynthesized from IAA-amino acids. IAA-N-Glc and/or its conjugates were also found in extracts of Arabidopsis, Lotus japonicus, and maize, suggesting that N-glucosylation of indole can be the common metabolic pathway of IAA in plants.     

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis

The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension–rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems. Received: 21 September 1998 / Accepted: 23 January 1999     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -     

Biosynthesis,conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana

Plant Molecular Biology -