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.     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Radioimmunoassay for determination of indole-3-acetic acid in fungi and plants

A radioimmunoassay technique for indole-3-acetic acid is described. The method has successfully been used to measure extractable indole-3-acetic acid in fungal and plant materials and is able to detect as little as 0.3 pmol. As non-radioactive antigen the methyl ester of indole-3-acetic acid is used and the radioactive antigen is tritiated. An acid-catalyzed esterification of indole-3-acetic acid is used for conversion into methyl ester. The measuring range of the assay is 0.3–10 pmol. In the assay, separation of free and bound fractions is achieved by dextran-coated charcoal, leaving the bound fraction in the supernatant.     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

A fluorometric method for the kinetic assay of indole-3-acetic acid oxidase.

The oxidation of IAA by peroxidase (1) and by more specific oxidases (2) leads to the formation of products which may have physiological activity (3, 4). The colorimetric estimation of IAA oxidation products involving reaction with p-dimethylaminocinnamaldehyde (DMACA) is reported to be more sensitive than other end point determinations such as the Salkowski and Ehrlich procedures which monitor the disappearance of IAA (5). These methods are end point procedures and, as such, are awkward and time consuming and present difficulties in obtaining kinetic data and measuring lag times. IAA oxidation has also been monitored by measuring ¹⁴CO₂ released from [1-¹⁴C] IAA (6) and uv spectral shifts during oxidation of IAA were reported by Meudt (3). The present paper reports a new procedure for the assay of horseradish peroxidase catalyzed oxidation of IAA. The assay procedure is based on the continuous measurement of a fluorescent product of the reaction.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

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.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Short-term response of Pisum stem segments to indole-3-acetic acid

The short-term response of green pea stem segments to indole-3-aceticacid (IAA) was investigated by continuously recording stem elongationwith a differential transformer. Stem segment elongation promotedby IAA began following a latent period after application. Thelatent period was more effectively shortened by raising thetemperature rather than the concentration of IAA; it was reducednearly to 0 min by treatment at 40?C. The length of the latentperiod was not affected by turgor pressures of stem cells, thoughthe rate of stem growth was diminished at lower turgor pressures.Stems pretreated with actinomycin D for 60 min, cycloheximidefor 30 min or colchicin for 6 hr were similar to untreated stemsin their short term response to IAA. This implies that the initiallypromoted elongation does not result from the activity of enzymessynthesized during the latent period by the action of IAA. (Received April 5, 1973; )     

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.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.