L'acide indolyl-3-lactique et son métabolisme chez Rhizobium

Summary Among the indole compounds formed when tryptophan 2-¹⁴C is metabolized by Rhizobium, indole-3-lactic acid (ILA) is specially studied. In the course of experiments carried out in the culture medium of growing Rhizobium and in suspensions of washed bacterial cells the amount of ILA formed is compared with that of indole-3-acetic acid (IAA) occurring simulataneously. The formation of ILA and that of IAA directly depend on a transamination reaction. A large quantity of ILA is present in suspensions of washed bacterial cells.When ILA alone, as precursor, is incubated with Rhizobium, several products are identified: IAA, indole-3-acetaldehyde and tryptophol. Tryptophan is also detected in the aqueous fraction and is labelled when ILA 2-¹⁴C is used. The pathway of this metabolism are discussed and a general scheme is suggested.     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Metabolism of indole-3-acetaldoxime in plants

Summary Living tissues of diverse plants representing 17 families were infiltrated with indole-3-acetaldoxime (IAAld oxime) in phosphate buffer, pH 6, and incubated for 3 hours at 25°C. Indole compounds were then extracted, separated and identified by paper or thin-layer chromatography (TLC). Indole-3-acetic acid (IAA) was quantitatively determined. Every tissue tested converted the oxime to IAA and tryptophol (T-ol). While accumulation of indole-3-acetonitrile (IAN) was observed in the non-acidic fractions of extracts of tissues of 8 species, indole-3-acetaldehyde (IAAld) accumulated in only a single tissue viz. Amaranthus shoot.IAAld oxime undergoes spontaneous hydrolysis at pH values below 4.7 leading to the formation of IAAld. Ce l-free preparations of etiolated Avena coleoptiles appear to contain an enzyme system capable of hydrolysing the oxime to IAAld. In the presence of such preparations, more IAAld and IAA are formed at all tested durations than the spontaneously formed IAAld. In the presence of bisulfite or semicarbazide, no IAA is formed, suggesting the intermediary formation of IAAld. The compound trapped with sodium bisulfite resembles very closely synthetic IAAld in its IR spectrum.In intact tissues, therefore, IAAld oxime appears to be first hydrolysed to IAAld which is then partly oxidized to IAA and mostly reduced to T-ol. Besides other evidence, formation of T-ol in every instance is believed to indicate the intermediary formation of IAAld. The nitrile pathway is considered to be only of minor importance in normal IAA biogenesis in the majority of higher plants.     

Identification of the metabolites of Indole-3-acetic acid in growing hypocotyls of Lupinus albus

The products of indole-3-acetic acid (IAA) metabolism by incubating hypocotyl sections and decapitated seedlings of Lupinus albus were investigated. Single treatments using [1-¹⁴C]-IAA, [2-¹⁴C]-IAA or [5-³H]-IAA and double treatments using [1-¹⁴C]-IAA+[5-³H]-IAA were carried out. Extracts from treated plant material were analyzed by paper chromatography (PC), Thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). When hypocotyl sections were incubated in [2-¹⁴C]-IAA, several IAA decarboxylation products including indole-3-aldehyde (IA1), indole-3-methanol (IM), 3-hydroxymethyloxindole (HMOx), methyleneoxindole (MOx) and 3,3-bisindolylmethane (BIM) were detected in the 95% ethanol extract; a latter extraction with 1M NaOH rendered IAA, IM and BIM, suggesting that conjugated auxins were formed in addition to conjugated IM. In sections incubated with [1-¹⁴C]-IAA, the 1M NaOH extraction also produced IAA so confirming the formation of conjugated auxins. The same decarboxylation products and two conjugated auxins, indole-3-acetylaspartic acid (IAAsp) and 1-O-(indole-3-acetyl)--D-glucose (IAGlu), were detected in the acetonitrile extracts from decapitated seedlings treated with [5-³H]-IAA. After a double isotope treatment ([1-¹⁴C]-IAA+[5-³H]-IAA) of decapitated seedlings, the ratio ¹⁴C/³H measured in the HPLC fractions of the acetonitrile extracts confirmed the presence of decarboxylation products as well as conjugated auxins.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Two natural indole glucosinolates from Brassicaceae

Four indole glucosinolates were isolated from different cruciferous tissues and tissue cultures. After separation in the form of their desulfo derivatives, they were separated by HPLC and analysed by mass spectrometry. Besides 3-indolylmethylglucosinolate and 1-methoxy-3-indolylmethylglucosinolate, two new compounds were identified as 5-hydroxy-3-indolylmethylglucosinolate and 5-methoxy-3-indolylmethylglucosinolate, respectively. Feeding experiments revealed that [ring-2-¹⁴C]tryptophan was a precursor of all of these glucosinolates and that [Me-¹⁴C]methionine serves as the methyl donor of the methoxylated derivatives.     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Analysis of Indole-3-Acetic Acid Metabolites from Dalbergia dolichopetala by High Performance Liquid Chromatography-Mass Spectrometry

A mixture of [2-¹⁴C₁] and [¹³C₆]indole-3-acetic acid was applied to the cotyledons of 6-day-germinated seeds of “jacarandá do cerrado” (Dalbergia dolichopetala) and after 8 hours the seeds were extracted. Analysis of the fractionated extract by reversed-phase high performance liquid chromatography-radiocounting revealed the presence of five radiolabeled metabolite peaks (I-V). After further purification, the individual peaks of radioactivity were analyzed by combined high performance liquid chromatography-steel filter-fast atom bombardment-mass spectrometry. The metabolite fraction V was found to contain [¹⁴C₁, ¹³C₆]indole-3-acetylas-partic acid and unlabeled indole-3-acetylglutamic acid. Analysis of the metabolite fraction II revealed the presence of dioxindole-3-acetylaspartic acid and putative dioxindole-3-acetylglutamic acid as well as putative benzene ring-hydroxylated derivatives of oxindole-3-acetylaspartic acid and oxindole-3-acetylglutamic acid. There was no evidence of significant incorporation of label from [2′-¹⁴C₁] or [¹³C₆]indole-3-acetic acid into any of these conjugated indoles.     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

Citrus Tissue Culture : AUXINS IN RELATION TO ABSCISSION IN EXCISED PISTILS

An in vitro bioassay for chemicals that affect Citrus abscission was used to identify three inhibitors of stylar abscission in lemon pistil explants incubated on defined nutrient media. The three inhibitors (picloram, 4-chlorophenoxyacetic acid, and 3,5,6-trichloropyridine-2-oxyacetic acid) are all auxins, and the most potent of them (i.e. picloram) was found to be at least 10 times more active in the bioassay than 2,4-dichlorophenoxyacetic acid. Picloram (2 micromolar) also was shown to be effective in inhibiting stylar abscission in pistil explants from other Citrus cultivars such as mandarin, Valencia, and Washington navel oranges and grapefruit. To study the physiology of auxins active as abscission inhibitors versus inactive auxins in lemon pistils, the transport and metabolism of [1-¹⁴C]-2,4-dichlorophenoxyacetic acid was compared with that of [2-¹⁴C]indole-3-acetic acid, which is without effect in the bioassay over the range from 0.1-100 micromolar. Insignificant quantities of labeled indole-3-acetic acid and/or labeled derivatives were found to reach the presumptive zone of stylar abscission under the test conditions. Labeled 2,4-dichlorophenoxyacetic acid and/or labeled derivatives also were transported slowly through pistils, but some radioactivity could be detected in the stylar abscission zone as early as 24 hours after the start of incubation. Extensive conversion of [2-¹⁴C]indole-3-acetic acid to labeled compounds tentatively considered to be glycoside and cellulosic glucan derivatives was found with the use of solvent extraction methodology. A significantly smaller percentage of the radioactivity in pistils incubated on [1-¹⁴C]-2,4-dichlorophenoxyacetic acid was found in fractions corresponding to these derivatives. Both transport and metabolism appear to be important factors affecting the activity of auxins as abscission inhibitors in the bioassay.     

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.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

Phytochemical Studies on the Tobacco Alkaloids

Duplicate feeding experiments of dl-ornithine-2-¹⁴C to the excised tobacco root culture were made, and the radioactive nornicotine was isolated. Approximately two thirds of the radioactivity was located in the 2-position of the pyrrolidine of the nornicotine in these experiments. This fact indicates that there are two modes in nornicotine biosynthesis: exclusive incorporation to the C-2 and equal incorporation to C-2 and C-5 from C-2 of ornithine.

On the basis of this finding, biosynthetic route was discussed.

dl-Ornithine-2-¹⁴C, dl-methionine-¹⁴CH₃ and partially racemized l-nornicotine-2,5-¹⁴C were administered to aseptically grown excised roots (N. rustica var. Brasilia). Incorporation of their radioactivity to nicotine was compared. The extent of their radioactive incorporation to nicotine was high in the order of ornithine, methionine and nornicotine; incorporation of radioactivity of nornicotine to nicotine was extraordinarily low. ¹⁵N-Labeled nornicotine was also fed to the same materials and ¹⁵N distribution was examined. Most of ¹⁵N still remained in the nornicotine reisolated. Marked amounts of ¹⁵N were located in the ethanol-insoluble fraction, the amino acid fraction and the substances having chromatographic R_F value close to that of nicotine. Only small amount of ¹⁵N was incorporated to the isolated nicotine.

Nornicotine is generally accepted to be a direct precursor of nicotine in tobacco plants. From these findings, however, it can be said that the biosynthesis of nicotine can occur through other routes without going through nornicotine.     

Ethylene modification of an auxin pulse in cotton stem sections

The effect of ethylene on the basipetal movement of indole-3-acetic acid-1-¹⁴C through cotton stem sections (Gossypium hirsutum, L. var. Stoneville 213) was studied apart from processes involved in the uptake and exit of auxin by the section. Stem sections 60 mm in length were pretreated with ethylene or placed in room air (control) and pulse labeled for 20 min with IAA-1-¹⁴C. In both the ethylene treated and control sections, the IAA-1-¹⁴C taken up moved basipetally as a peak of radioactivity. Generally, the applied pulse moved down the stem sections at an average velocity of approximately 5.8 mm per hr. In some experiments, however, ethylene slightly reduced the velocity of auxin transport. Although the peak of radioactivity became broader and more dispersed during its migration through the section, it was still distinguishable after 7 hr of transport.