Double-standard isotope dilution assay: I. Quantitative assay of indole-3-acetic acid

Isotope dilution analysis for the quantitation of labile compounds has been limited in applicability by the amount of sample necessary to determine specific activity. A method is described for the analysis of radiolabeled compounds which allows the direct determination of specific activity by gas chromatography. It requires the availability of the radiolabeled internal standard, as is customarily used in an isotope dilution assay, and also requires a chemically related radiolabeled compound to serve as a second internal standard. It is this second internal standard, added in known amounts, that permits quantitation of the gas chromatography. The method is illustrated by assaying indole-3-acetic acid in plant extracts using [¹⁴C]indole-3-acetic acid as the internal standard and adding [¹⁴C]indole-3-butyric acid as the second internal standard for quantitation of the gas chromatographic procedures. Used with a nitrogen-specific thermionic detector the method is selective and is sensitive at the nanogram level. The synthesis of [2-ring-¹⁴C]indole-3-butyric acid is also described.     

Changes in indole-3-acetic acid,indole-3-acetic acid oxidase,and peroxidase isoenzymes in the seeds of developing peach fruits

Changes in indole-3-acetic acid (IAA) content of peach (Prunus persica L. Batsch cv. Merry) seeds were followed during fruit development. The highest concentration of IAA, 2.7 g/g fresh weight, was found at the beginning of Stage III of fruit development, approximately 50–60 days after anthesis. The IAA-decarboxylating capacity of crude extracts of seeds was also greatest at 55–60 days after anthesis. Four soluble peroxidase isoenzymes were found on anionic electrophoresis. There were no marked changes in two isoenzymes (R _f 0.23 and 0.51), which were present in all three stages of fruit growth. There was a marked increase in a band atR _f 0.59 between Stages II and III, and a decrease in a band atR _f 0.68 from Stages II to III. Neither band (R _f 0.59 and 0.68) was present at Stage I.     

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.     

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.     

A new fluorometric assay method for quinolinic acid

A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215–220°C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assay method for uinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assay method may be practically applicable.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

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.     

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.     

A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.     

Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires addition of the substrates for induction and is oxygen dependent. The highest activity is obtained when the concentration of inducer is 0.2 mM. Spectrophotometric data are consistent with the suggestion that the indole ring is broken during degradation of IAA. We hypothesize that the enzyme catalyzes an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation product in the proposed pathway.     

A specific radioimmunoassay for nanogram quantities of the auxin,indole-3-acetic acid

We have developed a specific radioimmunoassay [RIA] for indole-3-acetic acid (IAA) in the 0.2 ng to 12 ng range which, in principle, can be extended to other indole auxins as well. Methods are presented for obtaining suitable antibody, for the RIA procedure, and for measuring IAA in methanolic extracts of plant tissues. Antibody specific for IAA was obtained from rabbits immunized with IAA bound to bovine serum albumin by formaldehyde treatment. In assays with this antibody, 2,4-dichlorophenoxyacetic acid and indoles structurally related to IAA reacted from 300- to 3000-fold less than did IAA itself. However, -and -naphthaleneacetic acid reacted significantly and hence interfered with the assay. Extracts of tobacco (Nicotiana tabacum L.) tissue were immunoassayed after partial purification by buffer-ether partition. Crown-gall tumor tissue, which is auxin-autotrophic, and pith tissue depleted of auxin by the diffusion method contained, respectively, 26.7 ng and <0.5 ng extractable IAA per gram fresh weight.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - -NAA -naphthalenacetic acid - PBS phosphate-buffered saline - RIA radioimmunoassay     

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.     

Catabolism of indole-3-acetic acid and 4- and 5-chloroindole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid. Indoleacetic acid (IAA), 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA were metabolized to different extents by strains 61A24 and 110. Metabolites were isolated and analyzed by high-performance liquid chromatography and conventional mass spectrometry (MS) methods, including MS-mass spectroscopy, UV spectroscopy, and high-performance liquid chromatography-MS. The identified products indicate a novel metabolic pathway in which IAA is metabolized via dioxindole-3-acetic acid, dioxindole, isatin, and 2-aminophenyl glyoxylic acid (isatinic acid) to anthranilic acid, which is further metabolized. Degradation of 4-Cl-IAA apparently stops at the 4-Cl-dioxindole step in contrast to 5-Cl-IAA which is metabolized to 5-Cl-anthranilic 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.     

Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.     

The function of free carboxyl groups in the action of peroxidase and indole-3-acetic acid oxidase

The extracellular peroxidase from cultures of Inonotus radiatus and of peanut (Arachis hypogeaL.) cells as well as the mycelial peroxidase from Trametes versicolor were used for studies of immobilizing this protein either by its free amino or its carboxyl groups. The immobilization process was carried out either on keratin proteins derived from feathers or on polyamide coated over silica gel. Coupling was established either through the free amino or carboxyl groups. In general the indolyl-3-acetic acid oxidase activity of fungal peroxidases exceeds that of peanut peroxidase. When the peroxidase of the three sources was immobilized on the matrices by the free amino groups, little if any effect on the IAA oxidase activity could be measured. However, immobilization through the carboxyl groups resulted in a drastic reduction of indole-3-acetic acid oxidase activity. Since identical amounts of peroxidase were linked in all cases, the loss of indolyl-3-acetic acid oxidase activity implies that the carboxyl group is essential for the active site.