Enzymatic dehydration of 3-hydroxymethyloxindole

Crude and partially purified extracts of wheat (Triticum vulgare, red variety) germ catalyze the dehydration of 3-hydroxymethyloxindole to 3-methyleneoxindole. Examination of the ultraviolet absorption spectrum of a reaction mixture consisting of either the extract or partially purified enzyme and 3-hydroxymethyloxindole, shows that this oxindole has undergone complete dehydration to 3-methyleneoxindole. TPNH-linked 3-methyleneoxindole reductase, also a constituent of the wheat germ extract, can be separated from the dehydrase by passage through an Agarose 15 column. Utilizing these partially purified enzymes, it can be demonstrated that the dehydrase activity found in wheat germ is a discrete enzymatic function.     

Inhibitory oxidation products of indole-3-acetic Acid: 3-hydroxymethyloxindole and 3-methyleneoxindole as plant metabolites

Extracts of pea seedlings (Pisum sativum, variety Alaska) oxidize indole-3-acetic acid to a bacteriostatic compound which has been identified as 3-hydroxymethyloxindole. At physiological pH this compound is readily dehydrated to 3-methyleneoxindole, another bacteriostatic agent. The extracts of pea seedlings also contain a reduced triphosphopyridine nucleotide-linked enzyme which reduces 3-methyleneoxindole to 3-methyloxindole, a non-toxic compound.

These enzymatic reactions also take place in intact seedlings; thus, a pathway of indole-3-acetic acid degradation via oxindoles appears to be pertinent to plant metabolism.

The significance of such metabolism lies in the fact that a key intermediate of this pathway, 3-methyleneoxindole, is a sulfhydryl reagent capable of profound effects on metabolism and growth.

     

The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules

Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, an oxidation product of indole-3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from ¹⁴C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3-methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of ¹⁴C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3-methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules.     

Auxin activity of 3-methyleneoxindole in wheat

A product of the enzymatic oxidation of indole-3-acetic acid, 3-methyleneoxindole, is at least 50-fold more effective than indole-3-acetic acid in stimulating the growth of wheat (Triticum vulgare, red variety) coleoptiles. Ethylenediaminetetra-acetic acid can antagonize the growth-stimulating properties of the parent compound, indole-3-acetic acid, presumably by chelating Mn²⁺, which is required for the enzymatic oxidation of indole-3-acetic acid. The growth stimulating effect of 3-methyleneoxindole, a product of the blocked reaction, on the other hand, is still evident in the presence of ethylenedia-minetetraacetic acid. In the presence of 2-mercaptoethanol, indole-3-acetic acid fails to stimulate the elongation of wheat coleoptiles. The property of binding to sulfhydryl compounds including 2-mercaptoethanol is unique to 3-methyleneoxindole among indole-3-acetic acid and its oxidation products. These findings suggest that 3-methyleneoxindole is an obligatory intermediate in indole-3-acetic acid induced elongation of wheat coleoptiles.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Heat-Melt of β-1, 3-d-Glucan Gel

Six new products of oxidation of indolyl-3-acetic add catalyzed by horseradish peroxidase were isolated, along with four known ones, 3-hydroxymethyloxindole (1), 3-methyleneoxindole (2), indolyl-3-aldehyde (4), and 3,3-diindolylmethane (10). Based on spectroscopic and chemical evidence, the new products were identified as 3-acetoxyindole (3), 3-(indol-3-ylmethyl)oxindole (6), 3-[(2-mdol-3-ylmethyl)indol-3-ylmethyl]oxindole (9), the 3-hydroxymethyl compounds of 6 and 9 (5 and 7), and 2-(indol-3-ylmethyl)indolyl-3-acetic acid (8), respectively.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Studies on indoleacetic acid oxidation by liquid medium from crown gall tissue cells: The role of malic acid and related compounds

1.

1. Indoleacetic acid oxidation by liquid medium from crown gall tissue culture cells has been studied. The reaction has a pH optimum of 4.5 and requires Mn²⁺ and a monohydric phenol. A short lag phase is routinely observed. The appearance of peroxidase and indoleacetic acid oxidising activity in the medium of a tissue culture was followed over a 3 week time course. One function of this enzyme may be to prevent the accumulation of excess inhibitory concentrations of indoleacetic acid.     

Generation and suppression of microsomal ribonuclease activity after treatments with auxin and cytokinin

RNase activity was assayed in subcellular fractions of apical regions of Pisum sativum L. var. Alaska epicotyls after seedling decapitation and treatments with various growth regulators. High concentrations of applied indoleacetic acid caused a marked increase to occur in the RNase activity level associated with “heavy” microsomes, e.g., a 20-fold rise per unit RNA or protein in 3 days. This rise could be abolished by treating with the cytokinin benzyladenine along with indoleacetic acid. Nevertheless, indoleacetic acid and benzyladenine acted synergistically in their abilities to evoke swelling and net synthesis of RNA and protein. Polysomal profiles prepared after treatment with indoleacetic acid plus benzyladenine showed less degradation than profiles from any other treatment. It is concluded that auxin generates and cytokinin suppresses the activity of a particular membrane-bound RNase which can control turnover of the auxin-evoked polysomes required for growth in peas. Synergism between the two hormones in this system may be explained by the action of one to increase RNA synthesis and the other to decrease RNA destruction.     

Inactivity of 3-methyleneoxindole as mediator of auxin action on cell elongation

The recently reported growth-promoting ability of 3-methyl-eneoxindole was examined in order to test the hypothesis that indole-3-acetic acid acts as a growth promoter only after oxidative conversion to 3-methyleneoxindole. Methyleneoxindole was synthesized from indole-3-acetic acid and N-bromosuccinimide, and its identity was confirmed by ultraviolet absorption, infrared absorption, mass spectrometry, and melting point. Methyleneoxindole was found to lack growth-promoting activity in coleoptile and pea (Pisum sativum) stem segments. Chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid, was found to have no inhibitory effect on growth promotion by indole-3-acetic acid. It is concluded that 3-methyleneoxindole is inactive as a growth promoter and therefore does not mediate the action of auxin on cell elongation.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Enhancement of Enzymatic Synthesis of Citrate in vitro by Indole-3-Acetic Acid

Indoleacetic acid in physiological concentrations was shown to enhance the synthesis of citiate by purified citrate condensing enzyme from castor beans and pig heart. Michaelis constants reveal that with indoleacetic acid in the reaction mixture a higher concentration of acetyl-CoA was necessary to give maximal velocity. V values with indoleacetic acid in the reaction (physiological concentrations) exceeded V without indoleacetic acid in reaction. Citric acid synthesized from ¹⁴C acetyl CoA was highly radioactive when indoleacetie acid was present in the reaction, indicating that indoleacetic acid did in fact enhance the synthesis. The data were discussed from the point of view that these studies may provide the basis for studies directed at ultimate understanding of the mechanism of action of indoleacetic acid.     

Auxin Does Not Alter the Permeability of Pea Segments to Tritium-labeled Water

The possibility of an auxin effect on the permeability of pea (Pisum sativum L. ev. Alaska) segments to tritium-labeled water has been investigated by three separate laboratories, and the combined results are presented. We were unable to obtain any indication of a rapid effect of indoleacetic acid on the efflux of ³HHO when pea segments previously “loaded” for 90 minutes with ³HHO were transferred to unlabeled aqueous medium with indoleacetic acid. We were able to confirm that segments pretreated with ³HHO plus indoleacetic acid for 60 to 90 minutes can show an enhanced ³HHO release as compared with minus indoleacetic acid controls. However, this phenomenon appears to be due to an increased uptake of ³HHO during the prolonged indoleacetic acid pretreatment, and therefore we conclude that auxin does not alter the permeability of pea segments to ³HHO in either short term or long term tests. We confirm previous reports that the uptake of ³HHO in pea segments proceeds largely through the cut surfaces, and that the cuticle is a potent barrier to ³HHO flux.     

Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using ¹³C₆-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).     

Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro

Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The “survival” of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues.     

Effect of gibberellic Acid on elongation and longevity of coleus petioles

The effects of gibberellic acid on the longevity and elongation of variously aged, debladed petioles of Coleus blumei were studied, with particular reference to the hypotheses 1) that auxin increases longevity by increasing growth, and 2) that gibberellic acid acts by increasing the endogenous levels of auxin.

Gibberellic acid, substituted for the leaf blades, significantly decreased longevity of younger petioles, as measured by days or hours to abscission. Gibberellic acid also decreased the longevity resulting from 0.1% indoleacetic acid. This is the opposite of the effect expected if it is increasing auxin levels in the petiole.

In its effect on elongation of younger petioles, however, gibberellic acid did act in the direction expected if it were increasing effective levels of auxin in the petiole. The elongation rate from 0.1% gibberellic acid plus 0.1% indoleacetic acid in lanolin was as large or larger than that for 1.0% indoleacetic acid.

Petioles which were 10 or more weeks old (i.e., at positions 5+ below the apical bud were not affected by 0.1% gibberellic acid in either longevity or rate of elongation, with or without 0.1% indoleacetic acid. Since 1.0% indoleacetic acid increases both longevity and elongation rate of these petioles over 0.1% indoleacetic acid, gibberellic acid is clearly not acting on older petioles as if it were increasing effective auxin levels).

     

[3H]Indole-3-Acetyl-myo-Inositol Hydrolysis by Extracts of Zea mays L. Vegetative Tissue

[³H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37°C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as α-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other fraction enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.     

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl₂ and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [³H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.     

Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Changes in the pattern of protein synthesis induced by 3-indolylacetic Acid

Experiments have been performed to investigate whether indoleacetic acid changes the balance between the rates of synthesis of different kinds of proteins. Sub-apical sections of etiolated peas were incubated with ¹⁴C- or ³H-labeled amino acid, and combined to give dual-labeled tissue. Cell fractions were prepared by differential centrifugation, and the dual-labeled protein of each fraction analyzed by gel-filtration. When 2 × 10⁻⁵ m indoleacetic acid was included with ¹⁴C-labeled amino acid, but not with the ³H-labeled amino acid, pronounced changes occurred in the pattern of incorporation of the ¹⁴C label into protein. These changes were greatest in the proteins of the particulate fraction which included nuclear material. Although the pattern of incorporation of lysine was shown to be different from that of leucine, the changes induced by indoleacetic acid were quantitatively similar whichever amino acid was used as a precursor. Dual-labeled protein was further fractionated using column chromatography on DEAE-cellulose. The results suggested that the effect of indoleacetic acid may not be completely general, and that the pattern of synthesis of many proteins may be unaltered by indoleacetic acid. When tissue was preincubated with 10 μg/ml actinomycin D for 30 minutes, incorporation of amino acid into protein was reduced but not abolished. Actinomycin D did, however, prevent the changes in the pattern of protein synthesis which were induced by indoleacetic acid.