Oxidation of Indole-3-Acetic Acid to Oxindole-3-Acetic Acid by an Enzyme Preparation from Zea mays

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.     

Movement of Indole-3-acetic Acid and Tryptophan-derived Indole-3-acetic Acid from the Endosperm to the Shoot of Zea mays L

The structures and the concentrations of all of the indolylic compounds that occur in the endosperm of the seeds of corn (Zea mays L.) are known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the endosperm can be transported to the seedling in significant amounts and thus help identify the seed-auxin precursor of Cholodny (1935. Planta 23:289-312) and Skoog (1937. J. Gen. Physiol. 20:311-334). Of interest is the transport of tryptophan, indole-3-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds of the seed. We have shown that: (a) IAA can move from the endosperm to the shoot; (b) the rate of movement of IAA from endosperm to shoot is that of simple diffusion; (c) 98% of the transported IAA is converted into compounds other than IAA, or IAA esters, en route; (d) some of the IAA that has moved into the shoot has been esterified; (e) labeled tryptophan applied to the endosperm can be found as labeled IAA in the shoot; and (f) with certain assumptions concerning IAA turnover, the rate of movement of IAA and tryptophan-derived IAA from the endosperm to shoot is inadequate for shoot growth or to maintain IAA levels in the shoot.     

Measurement of the Rates of Oxindole-3-Acetic Acid Turnover, and Indole-3-Acetic Acid Oxidation in Zea mays Seedlings

Nonhcbcl, H. M. 1986. Measurement of the rates of oxindole-3-aceticacid turnover and indole-3-acetic acid oxidation in Zea maysseedlings.—J. exp. Bat. 37: 1691–1697. Oxindole-3-acetic acid is the pnncipal catabolite of indole-3-aceticacid in Zea mays seedlings. In this paper measurements of theturnover of oxindole-3-acetic acid are presented and used tocalculate the rate of indole-3-acetic acid oxidation. [³H]Oxindolc-3-acetic acid was applied to the endosperm of Zeamays seedlings and allowed to equilibrate for 24 h before thestart of the experiment. The subsequent decrease in its specificactivity was used to calculate the turnover rate. The averagehalf-life of oxindole-3-acetic acid in the shoots was foundto be 30 h while that in the kernels had an average half-lifeof 35 h. Using previously published values of the pool sizesof oxindole-3-acetic acid in shoots and kernels from seedlingsof the same age and variety, and grown under the same conditions,the rate of indole-3-acetic acid oxidation was calculated tobe I-I pmol plant^–1 h^–1 in the shoots and 7·1pmol plant^–1 h^–1 in the kernels. Key words: Oxindole-3-acetic acid, indole-3-acetic acid, turnover, Zea mays     

Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L

Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [¹⁴C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified.     

Analysis of Indole-3-acetic Acid Metabolism in Zea mays Using Deuterium Oxide as a Tracer

A method using deuterium oxide (D₂O) as a tracer was used to study indole-3-acetic acid (IAA) metabolism in Zea mays seedlings. Seeds were imbibed and grown for 4 days in 30% D₂O in the dark. IAA was then isolated from roots and shoots and analyzed for deuterium content by mass spectrometry. We found that a significant portion of the IAA isolated from plants had incorporated deuterium at nonexchangeable sites of the indole ring. This indicates that some of the IAA in the germinating seedling is made via de novo indole synthesis. Moreover, we found that the deuterium content of IAA was 2.6 times greater in shoots than in roots. These results indicate that at least some of the IAA in roots and shoots came from different biosynthetic pathways. It appears that the fraction of IAA produced via de novo indole synthesis is greater in shoots than in roots.     

Myo-Inositol Esters of Indole-3-acetic Acid Are Endogenous Components of Zea mays L. Shoot Tissue

Indole-3-acetyl-myo-inositol esters have been demonstrated to be endogenous components of etiolated Zea mays shoots tissue. This was accomplished by comparison of the putative compounds with authentic, synthetic esters. The properties compared were liquid and gas-liquid chromatographic retention times and the 70-ev mass spectral fragmentation pattern of the pentaacetyl derivative. The amount of indole-3-acetyl-myo-inositol esters in the shoots was determined to be 74 nanomoles per kilogram fresh weight as measured by isotope dilution, accounting for 19% of the ester indole-3-acetic acid of the shoot. This work is the first characterization of an ester conjugate of indole-3-acetic acid from vegetative shoot tissue using multiple chromatographic properties and mass spectral identification. The kernel and the seedling shoot both contain indole-3-acetyl-myo-inositol esters, and these esters comprise approximately the same percentage of the total ester content of the kernel and of the shoot.     

Effect of Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots of Zea mays Seedlings

We wished to determine the effect of endosperm removal on the amounts of free and esterified indole-3-acetic acid (IAA) in young Zea mays seedlings. The increases of IAA derived from endosperm and from biosynthesis, but without correction for catabolic losses, were 0.9 picomole of free IAA per shoot per hour, and 1.1 picomoles per shoot per hour of ester IAA. After deseeding, free IAA in the shoot declines by 40% following kernel removal and total (free + ester) IAA declines at a rate of about 1 picomole per shoot per hour. A slight, but insignificant increase of ester IAA occurs following endosperm removal. In the primary roots, the decreases of free IAA and total (free + ester) IAA are accelerated by seed removal. Thus, the endosperm appears to be a major source of IAA for the shoot and root.     

Concentrations of Indole-3-acetic Acid and Its Esters in Avena and Zea

An isotope-dilution method has been developed for the assay of free indole-3-acetic acid and ester indole-3-acetic acid as measured by indole-3-acetic acid liberated by mild alkaline hydrolysis. Application of this method to seedlings of Avena sativa and Zea mays indicates the upper limit of free indole-3-acetic acid in Avena to be about 16 μg per kg and in Zea, about 24 μg. The amount of 1 n alkali-labile indole-3-acetic acid in Zea is about 330 μg per kg and there is very little 1 n alkali-labile IAA in Avena. A chemical characterization of the indole-3-acetic acid of Avena and a confirmation of the chemical characterization of the indole-3-acetic acid of Zea is presented.     

Effect of Endosperm Removal on 7 Normal NaOH-Labile Indole-3-acetic Acid Conjugates in Shoots and Roots of Zea mays Seedlings

The pool of amide-linked indole-3-acetic acid (amide IAA) in the shoot of growing etiolated seedlings of Zea mays increases between the 3rd and 5th day of germination to equal the amount of free IAA and two-thirds the amount of ester IAA. Deseeding the germinant changes the pool size of free and amide IAA in a manner suggestive of conversion of endogenous free IAA to amide IAA. Deseeding also caused an almost total disappearance of amide IAA from the root, demonstrating that the pool of amide IAA is not inert and can be actively metabolized in young Z. mays seedlings.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Indole-3-acetic Acid Levels and the Role of Indole-3-acetic Acid Oxidase in the Normal Root and Club-root of Cabbage

The auxin content of club-root (Plasmodiophora brassicae Wor.) is 50–100 times higher than that of normal cabbage root. The importance of this difference in the disease development is discussed. Both normal root and club-root of cabbage contain allosteric IAA oxidase and IAA oxidase with ordinary kinetic properties. In normal cabbage root the allosteric one is associated with cell fractions sedimenting at 20,000 × g and 105,000 × g, in club-root it remains in the supernatant after 105,000 × g centrifugation. IAA oxidase with conventional kinetic properties is present in both these tissues in the cell fraction sedimenting at 10,000 × g, which contains mainly cell wall fragments. It is concluded that IAA oxidase is not primarily involved in regulation of the endogenous IAA level.     

Antioxidative Activities of Oxindole-3-acetic Acid Derivatives from Supersweet Corn Powder

The components contributing to the antioxidative activity of supersweet corn powder (SSCP), which is commonly used in corn soup and snacks in Japan, were clarified and the effects investigated. 7-(O-β-Glucosyloxy)oxindole-3-acetic acid (GOA) was found to be the component most strongly contributing to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of the 80% ethanol extract of SSCP, and the presence of its aglycone, 7-hydroxy-oxindole-3-acetic acid (HOA) was confirmed. GOA and HOA respectively contributed 35.1% and 10.5% to the DPPH radical-scavenging activity of the 80% ethanol extract of SSCP. Mice orally administered with HOA at doses of both 500 and 1500 mg/kg showed a significantly lower (p<0.05) level of thiobarbituric acid reactive substances (TBARS) in the plasma than the vehicle-treated control. These results suggest that GOA and HOA were at least partly involved in the antioxidative activity of SSCP in vitro and that HOA might have possessed antioxidative activity in vivo.     

Oxidative Reaction of Oxindole-3-acetic Acids

The oxindole-3-acetic acids, oxidative metabolites of indole-3-acetic acid, were isolated from a byproduct of a corn starch manufacturing plant, and were further converted to the 3-hydroxyl derivatives in the presence of metal ion. The mechanical study was followed by a chemical analysis including other byproducts, and suggested the presence of an intermediate that had a radical at the C-3 position of oxindole-3-acetic acids.     

Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.)

Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [¹⁴C]IAA and [¹³C₆]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The K_m value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant.     

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.     

Transport and Metabolism of Indole-3-Acetyl-myo-Inositol-Galactoside in Seedlings of Zea mays

Indole-3-acetyl-myo-inositol galactoside labeled with ³H in the indole and ¹⁴C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [³H]indole-3-acetyl-myo-inositol and [³H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumptions concerning the equilibration of applied [³H]indole-3-acetyl-myo-inositol-[U-¹⁴C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [³H]indole-3-acetyl-myo-inositol-[¹⁴C] galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [³H]indole-3-acetyl-myo-inositol-[¹⁴C]galactose supplies appreciable amounts of ¹⁴C to the shoot and both ¹⁴C and ³H to an uncharacterized insoluble fraction of the endosperm.