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.     

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.     

Mechanism of a Synergistic Effect of Kinetin on Auxin-induced Ethylene Production: Suppression of Auxin Conjugation

In hypocotyl segments of mung bean (Phaseolus mungo L.) seedlings, exogenously supplied indoleacetic acid was rapidly conjugated mainly into indoleacetylaspartic acid, which was inactive in inducing ethylene production. Kinetin is known to stimulate indoleacetic acid-induced ethylene production. The mechanism of kinetin action on indoleacetic acid-induced ethylene production by hypocotyl segments of mung bean seedlings was studied in relation to indoleacetic acid uptake and indoleacetic acid metabolism. Kinetin enhanced indoleacetic acid uptake during the initial 2-hour incubation and markedly suppressed the conversion of indoleacetic acid to indoleacetic acid conjugates throughout the whole 7-hour incubation. As a result, there was more free indoleacetic acid and less conjugated indoleacetic acid in the segments treated with kinetin than in those receiving no kinetin. A close relationship was demonstrated between the rate of ethylene production and the level of free indoleacetic acid, which was regulated by kinetin.     

Peroxide Levels and the Activities of Catalase, Peroxidase, and Indoleacetic Acid Oxidase during and after Chilling Cucumber Seedlings

The activities of catalase, peroxidase, indoleacetic acid (IAA) oxidase and peroxide levels in cucumber plants during and after chilling were determined. During 96 hours at 5 C and 85% relative humidity, catalase activity declined, IAA oxidase activity increased, and peroxide concentrations increased. Peroxidase activity was not affected by chilling. When chilled plants were returned to 25 C to recover, enzyme activities and peroxide concentration were restored to their prechilling levels. The increase in peroxide and IAA oxidase activity may inactivate or destroy IAA and thus retard growth.     

Response of Substituted Indoleacetic Acids in the Indolo-α-pyrone Fluorescence Determination

The method of indolo-α-pyrone fluorescence-determination of indoleacetic acid was investigated to study possible interference from 4-chloro-indoleacetic acid and 5-hydroxyindoleacetic acid, which occur naturally. Both compounds show about 40% of the fluorescence of indoleacetic acid after conversion into their a-pyrones. Other halogenated indoleacetic acids show between zero and 60% of the fluorescence of indoleacetic acid. It is concluded that the concentration of indoleacetic acid cannot be determined in crude extracts in the presence of 4-chloro- or 5-hydroxy-indoleacetic acid, because separate determinations of each of these compounds are not possible by changing the excitation or fluorescence wave-lengths of the testing equipment.     

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.     

Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.     

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).

     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Turnover of cell wall polysaccharides in elongating pea stem segments

Turnover of cell wall polysaccharides and effects of auxin thereon were examined after prelabeling polysaccharides by feeding pea (Pisum sativum var. Alaska) stem segments ¹⁴C-glucose, then keeping the tissue 7 hours in unlabeled glucose with or without indoleacetic acid. There followed an extraction, hydrolysis, and chromatography procedure by which labeled monosaccharides and uronic acids were released and separated with consistently high recovery. Most wall polymers, including galacturonan and cellulose, did not undergo appreciable turnover. About 20% turnover of starch, which normally contaminates cell wall preparations but which was removed by a preliminary step in this procedure, occurred in 7 hours. Quantitatively, the principal wall polymer turnover process observed was a 50% decrease in galactose in the pectinase-extractable fraction, including galactose attached to a pectinase-resistant rhamnogalacturonan. Other pectinase-resistant galactan(s) did not undergo turnover. No turnover was observed in arabinans, but a doubling of radioactivity in arabinose of the pectinase-resistant, hot-acid-degradable fraction occurred in 7 hours, possibly indicating conversion of galactan into arabinan. None of the above changes was affected by indoleacetic acid, but a quantitatively minor turnover of a pectinase-degradable xyloglucan was found to be consistently promoted by indole-acetic acid. This was accompanied by a reciprocal increase in water-soluble xyloglucan, suggesting that indoleacetic acid induces conversion of wall xyloglucan from insoluble to water-soluble form. The results indicate a highly selective pattern of wall turnover processes with an even more specific influence of auxin.     

A red-far red reversible effect on uptake of exogenous indoleacetic Acid in etiolated rice coleoptiles

The uptake and accumulation of exogenous indoleacetic acid-¹⁴C by intact rice coleoptiles were examined. The absorption of exogenous indoleacetic acid was controlled by phytochrome, while the subsequent accumulation of this indoleacetic acid in various portions of the coleoptile was complex, and the effect of red light in this system was small compared to the alteration of the uptake of indoleacetic acid by red light. The absorption of indoleacetic acid exhibited two phases: the first occurring during the first 3-hour portion of the incubation was an inhibition, while the second was a promotive effect at about the 5th hour of incubation. Both of these effects were red, far redreversible, implicating phytochrome in this effect. Neither the destruction nor the immobilization of this exogenous indoleacetic acid apeared to be greatly affected by red light irradiation. The principal interaction between phytochrome and indoleacetic acid appears to occur during the absorption of exogenous indoleacetic acid. This effect may be related to the control by phytochrome of the amount of auxin which diffuses from coleoptile tips.     

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.     

The direct involvement of hydrogen peroxide in indoleacetic acid inactivation

Hydrogen peroxide appears to mask the chemical characteristics of indoleacetic acid. This was demonstrated by the Salkowski and Fluorescence tests. Stem elongation and root initiation were inhibited as a result of adding H₂O₂ to nutrient media containing IAA, however, upon the addition of purified catalase, most of the symptoms of IAA inactivation were reversed. It is suggested that in vivo IAA may be regulated partially by its conjugation with H₂O₂, and catalase may have a role in the IAA reactivation process. The accumulation of hydrogen peroxide in the cells as a result of catalase inhibition may lead to a temporary IAA inactivation, therefore effecting plant growth.     

The role of indoleacetaldehyde in IAA production from tryptophan by plants and by their epiphytic bacteria

Tryptophan, tryptamine, or indolepyruvic acid were applied to 2 systems: a bacterial (pea stem sections containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In the plant system, the production of indoleacetic acid and indoleethanol (tryptophol) from each applied indole derivative is clearly reduced by the aldehyde reagents bisulfite and dimedon, respectively. Indoleacetaldehyde is chromatographically detected after alkaline liberation from its bisulfite addition product. In the bacterial system, the production of indoleacetic acid and indoleethanol is likewise reduced by bisulfite and dimedon. However, after tryptophan or tryptamine application, we could not detect indoleacetaldehyde in the described way. In one case only, namely tryptamine application to the bacterial system, indoleethanol production (contrary to indoleacetic acid production) is scarcely reduced by the aldehyde reagents. This indicates a bacterial pathway tryptamine → indoleethanol which bypasses indoleacetaldehyde.     

The relationship of the peroxidative indoleacetic Acid oxidase system to in vivo ethylene synthesis in cotton

Since peroxidase and manganese have been implicated in both auxin destruction and ethylene production, the effect of auxins and high tissue levels of manganese on the peroxidative indoleacetic acid oxidase system and the internal level of ethylene was determined in cotton (Gossypium hirsutum L. cv. Watson GL-7). The highest level of manganese tested produced manganese toxicity symptoms, including necrotic lesions, accompanied by an increase in internal ethylene levels at about 15 days after treatment initiation. Statistically significant increases in indoleacetic acid oxidase and peroxidase activity were first observed 2 days later and were paralleled by tissue manganese levels above 7.4 milligrams per gram dry weight and internal ethylene levels of 0.77 microliters per liter air. Eight hours after application of 2,4-dichlorophenoxyacetic acid or indoleacetic acid, the internal levels of ethylene were increased to above 6.6 microliters per liter air in cotton plants, and levels of this magnitude were maintained for a 72-hour period of observation. Modification of peroxidase and indoleacetic acid oxidase activity in auxintreated plants definitely occurred well after the elevation of internal ethylene levels. While ethylene levels and indoleacetic acid oxidase activity were increased by both experimental approaches, the earlier appearance of increased ethylene indicates that the peroxidative indoleacetic acid oxidase system in cotton is not involved in ethylene synthesis or that this enzyme is not the rate-limiting factor when ethylene synthesis is increased. Ethylene, as well as auxin destruction, may be involved in some of the long term plant responses to toxic levels of manganese. The findings also suggest that auxin-induced ethylene may play a role in the elevation of peroxidase and indoleacetic acid oxidase activity eventually seen in extracts of plants treated with auxins. The data support the assumption that the enzymatic portion of the indoleacetic acid oxidase system in cotton is a peroxidase.     

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments.     

pH-dependency of Escherichia coli catalase activity under modified culture conditions

In the previous work we have found two peaks of catalase activity at acid and neutral pH in partially destroyed bacteria E. coli K12 KS400. The present study indicates that catalase activity with two pH-optimums is sensitive to pH of cultivation medium. The relative catalase activity of frozen-thawed bacteria preparations measured at pH 3.5 increased two-fold and activity measured at pH 7.0 didn't change by shift of medium pH from value 5.5 to 7.0. In analogical preparations of bacteria grown in slightly alkaline media activity with acid maximum was not observed, but activity with neutral maximum rose to 130% in comparison with the intact cells was revealed. Two peaks of activity differed in their sensitivity to bacteria destruction, heating, inhibition by NaN3 and AMT, oxidative stress. The analysis of recent literature information and experimental data leads us to conclude that the activity with neutral pH-optimum consists of two known catalase forms HPI and HPII in E. coli. The ratio of HPI and HPII is 70 and 30%, respectively what was concluded from inhibition of catalase activity with neutral pH-optimum by AMT. Properties of catalase activity with acid pH-optimum didn't corresponding to any known enzyme forms. It is suggested the activity measured at pH 3.5 is results of some unstable activator which acts in acid pH range. It is possible that the described activity with acid pH-optimum is specific for the used E. coli strain. Investigation of another strain of E. coli K12 AB1157 confirmed this idea where the activity peak with acid pH-optimum was not detected.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

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.     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.