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.     

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.     

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: II. Metabolic Requirements

The 2- to 4-fold rise in particle-bound β-glucan synthetase (uridine diphosphate-glucose: β-1, 4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca²⁺ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by α, α′-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.     

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 μm and 10 μm). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 μm) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 μm) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.     

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.     

Regulation of 3-indoleacetic acid production in Pseudomonas syringae pv. savastanoi. Purification and properties of tryptophan 2-monooxygenase

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.     

Indoleacetic acid stimulates cellulose deposition and selectively enhances certain β-glucan synthetase activities

Particulate preparations from growing regions of 8-day old Pisum sativum epicotyls catalysed glucosyl transfer to β-glucan from UDPglucose and GDP-glucose. The activities assayed with GDPglucose (6 or 600 μM) or low (6μM) concentrations of UDPglucose disappeared from decapitated epicotyls within 3 days, but were maintained when the cut apex was treated with the hormone indoleacetic acid. These activities re-appeared when indoleacetic acid was added 3 days after decapotation; cycloheximide prevented this response. The activity assayed with high (600 μM) concentrations of UDPglucose, in contrast, remained in the decapitated epicotyl unaffected by indoleacetic acid or cycloheximide during incubation periods of upt to 5 days. In competition experiments with the two substrates, the individual synthetase activities were not additive, and part of the activity with one substrate was still detectable in the presence of a large excess of the other.These observations indicate the existence in pea particles of at least 4 glucan synthetase activities which differ in substrate affinities, stability and developmental responses to treatments that affect growth and protein synthesis. Such treatments alo markedly influence the deposition of cellulose, e.g. indoleacetic acid caused an 8-fold increase in cellulose laid down in a 3-day period. It is suggested that indoleacetic acid-regulated synthetase activities account for the extra cellulose evoked by indoleacetic acid during sustained growth, and a different non-regulated synthetase activity is responsible for a basal rate of cellulose deposition which proceeds in the presence or absence 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.     

Hydroxyproline-rich cell wall protein (extensin): role in the cessation of elongation in excised pea epicotyls

The synthesis and accumulation of cell wall hydroxyproline increases coincident with the cessation of elongation growth in pea epicotyls. We examined the relationship between these biochemical and physiological events by using epicotyl sections challenged with α, α′-dipyridyl. This chelator blocked hydroxyproline biosynthesis without affecting overall protein synthesis. Epicotyl sections mimicked elongation growth in situ when placed in indoleacetic acid. Elongation was blocked by the addition of benzimidazole or Ethrel. These latter compounds acted independently as judged by their kinetics of action and the inhibition of Ethrel's effect only by CO₂.During rapid elongation growth in indoleacetic acid, there was no increase in cell wall hydroxyproline. However, incubation in either growth-inhibitory agent increased hydroxyproline 3-fold. When this increase was blocked by dipyridyl incubation, growth was not inhibited in benzimidazole or Ethrel, but proceeded at the maximal rate. During long-term incubations in buffer, cell wall hydroxyproline increased and the sections eventually became unable to grow. However, if dipyridyl was added to block the hydroxyproline increase, growth potential remained. Elongation was inhibited by supraoptimal concentrations of indoleacetic acid. However, such inhibition did not occur in the presence of dipyridyl.These results indicate that an hydroxyproline-containing component is necessary in rendering the cell wall inextensible when elongation growth ceases.     

Effects of Pargyline on Mitochondrial Amine Oxidase Activity, Indoleacetic Acid Synthesis and Growth of Crown-Gall Tumor Callus

Vinca rosea L. crown-gall tumor callus tissue cultures treated with N-benzyl-N methyl propargylamine (pargyline) exhibited a decrease in the level of endogenous indoleacetic acid from 0.42 μg/mg of protein to less than 0.30 μg/mg of protein. A simultaneous decrease in the specific activity of mitochondrial amine oxidase from 3000 units to less than 250 units at 1.0 μM, 0.01 mM, 0.1 mM and 1.0 mM pargyline, suggested a relationship between amine oxidase function and indoleacetic acid synthesis. Tryptamine incorporation into indoleacetic acid was also decreased at these concentrations. Pargyline inhibited tumor callus growth significantly (based on fresh weight measurements) at the highest concentration, 1.0mM. These data support the hypothesis of a coordinate metabolic system linking mitochondrial amine oxidase activity and indole acetic acid synthesis. Inhibitory action of pargyline on the enzyme is reflected in reduced indoleacetic acid levels and, ultimately, in reduced callus growth rates.     

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.     

Promotion of indoleacetic Acid oxidase isoenzymes in tobacco callus cultures by indoleacetic Acid

Indoleacetic acid oxidase in tobacco callus cultures (Nicotiana tabacum L., cv. White Gold) was composed of at least two groups of isoenzymes, which were distinctly different in electrophoretic mobilities and in responses to growth substances. Indoleacetic acid had dual effects; at low concentrations it promoted the development of two fast-migrating indoleacetic acid oxidase isoenzymes, but at high concentrations it increased the level of other indoleacetic acid oxidase isoenzymes with low and moderate electrophoretic mobilities. However, indoleacetic acid was not unique in such effects; 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were effective at concentrations lower than that of indoleacetic acid.     

Metabolism of Indole-3-Acetic Acid: II. Oxindole Pathway in Parthenocissus tricuspidata Crown-Gall Tissue Cultures

An indoleacetic acid oxidase preparation from an acetone powder of Parthenocissus tricuspidata crown-gall tissue has been examined. An intermediate in the reaction is 3-hydroxymethyloxindole and nonenzymic conversion of it to 3-methyleneoxindole was observed. Neither reaction mixtures nor 3-methyleneoxindole have any auxin-like activity in Avena or wheat coleoptile bioassays. In vivo studies show that although 53% decarboxylation of indoleacetic acid was observed in 48 hours, only a small amount of 3-methyloxindole could be recovered from the medium. The other decarboxylated products remain to be identified but are not 3-hydroxymethyloxindole or 3-methyleneoxindole.     

ENDOGENOUS BETA IRRADIATION AND TUMOR PRODUCTION IN AN AMPHIPLOID NICOTIAN A HYBRID

The amphiploid Nicotiana suaveolens × N. langsdorffii, which is genetically constituted to produce tumors spontaneously late in development, can be induced to form them in the early seedling stage by treatment of the apical meristem with kinetin and indoleacetic acid. Application of H³-or C¹⁴-labeled plant-growth regulators resulted in most plants and notably with C¹⁴-labeled indoleacetic acid in a significant increase in the rate of tumor production over growth-regulator treatment alone. Endogenous radiation alone was shown to be tumefacient since radioactive nucleosides, tritiated water, and C¹⁴-sodium carbonate also enhance tumorization.     

Endogenous and Dietary Indoles: A Class of Antioxidants and Radical Scavengers in the ABTS Assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS^?+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like l-tryptophan and derivatives (N-acetyltryptophan, l-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-β-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9?mM, usually higher than that for Trolox and ascorbic acid (1?mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS^?+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic β-carbolines (pyridoindoles), that did not scavenge ABTS^?+. Radical scavenger activity of indoles against ABTS^?+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Morphogenesis in Selaginella: II. Auxin Transport in the Root (Rhizophore)

The rhizophore of Selaginella willdenovii Baker develops from the ventral angle meristem. The morphological nature of this organ has been in dispute. The purpose of this investigation was to obtain physiological evidence to support the contention that the rhizophore is a root and not a shoot. This was accomplished by studying the movement of ³H-indoleacetic acid and ¹⁴C-indoleacetic acid in Selaginella rhizophores. In 6-millimeter tissue segments, twice as much radioactivity accumulated in acropetal receivers as in basipetal. During 1 hour of transport in intact roots auxin traveled twice as far in the acropetal direction as basipetal. A significant amount of radioactivity transported in the tissue was found to co-chromatograph with cold indoleacetic acid. Decarboxylation accounted for 10% loss of activity from donors. The data provide sufficient physiological evidence that this organ is morphogenetically a root.     

Conversion of indole-3-ethanol to indole-3-acetic Acid in cucumber seedling shoots

Indoleethanol-¹⁴C was applied to intact cucumber seedlings and to hypocotyl segments. The presence of indoleacetic acid-¹⁴C in tissue extracts was demonstrated by thin layer radiochromatography. There was no evidence of conversion of indoleacetic acid to indoleethanol. It is suggested that the growth-promoting activity of indoleethanol is due to its conversion to indoleacetic acid.     

Phosphorylated and nucleotide sugar metabolism in relation to cell wall production in Avena coleoptiles treated with fluroride and peroxyacetyl nitrate

Coleoptile sections of Avena sativa L. were pretreated with sodium fluoride or peroxyacetyl nitrate at levels which inhibit auxin-induced growth but did not affect glucose uptake or CO₂ production when postincubated for 30 minutes in a ¹⁴C-glucose medium without auxin. Labeling of metabolites involved in cell wall synthesis was measured. Peroxyacetyl nitrate decreased labeling, and it was concluded that the pool size of uridine di-phosphoglucose, sucrose, and cell wall polysaccharides decreased compared to control. The changes suggest that peroxyacetyl nitrate inactivated sucrose and cell wall synthesizing enzymes including cellulose synthetase and decreased cell growth by inhibiting production of cell wall constituents. Fluoride treatment had no effect on production of cell wall polysaccharides, with or without indoleacetic acid stimulation of growth. The only change after fluoride treatment was a decrease in uridine diphosphoglucose during incubation without indoleacetic acid, a decrease that disappeared when indoleacetic acid was present. It was concluded that some other aspect of cell wall metabolism, not determined here, was involved in fluoride-induced inhibition of growth.     

Geotropic curvature of decapitated Avena coleoptiles after application of tips of maize roots,tips of Avena coleoptiles,indoleacetic acid,or abscisic acid

Isolated Avena coleoptiles were decapitated at different distances from the tip and then placed horizontally, after which the geotropic curvature was measured. No geotropic curvature could be detected during the first 3 h. Later, upward curvature occurred which was found to depend inversely on the length of the decapitated tips. When the tips of maize roots or Avena coleoptiles were placed on the cut surface of decapitated Avena coleoptiles, the coleoptiles showed a significantly stronger upward curvature as compared to controls which had been provided with agar blocks on the cut surface. The same upward curvature was found with decapitated coleoptiles provided with agar blocks containing 10^-6 or 10^-7 M indoleacetic acid (IAA). After application of abscisic acid (ABA) at concentrations of 10^-6 and 10^-8 M to the decapitated coleoptiles, the curvature observed was not different from that of the controls; at higher concentrations of ABA the curvature was found to be lower than that of the controls. It is concluded that root tips secrete a substance which may replace the effect of IAA in coleoptiles. The results are discussed in view of the validity of the Cholodny-Went hypothesis for the geotropic reaction of roots.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles

The influence of indoleacetic acid, 0.03% CO₂, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO₂ stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO₂. Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO₂, and that indoleacetic acid increased the incorporation of ¹⁴C-bicarbonate into protein. These data are discussed in the context of CO₂-stimulated growth of etiolated tissue, and proposals that CO₂-stimulated growth involves dark CO₂ fixation.