Metabolism of 1-naphthaleneacetic acid in explants of tobacco: Evidence for release of free hormone from conjugates

1-Naphthaleneacetic acid (1-NAA), required for in vitro flower bud formation, was taken up by pedicel explants of tobacco (Nicotiana tabacum L.) in large amounts and rapidly metabolized into various conjugates. These conjugates have been tentatively identified in four thin-layer Chromatographic systems using authentic standards as references. The major metabolite formed during the first hours of culture comigrated with 1-NAA-glucoside (1-NAGlu). From the 6th hour on, most 1-NAA had been converted into a yet unidentified metabolite. 1-NAglu was an intermediate in the formation of this metabolite. After 24 h, 1-NAA-aspartate (1-NAAsp) became the second major metabolite. The increase in 1-NAAsp formation was induced by 1-NAA. The inactive analog 2-naphthaleneacetic acid (2-NAA) was metabolized similar to 1-NAA, but was unable to increase the formation of the aspartate conjugate. When explants were fed labeled 1-NAGlu, 1-NAAsp or the major unidentified metabolite, radioactivity became associated with free 1-NAA and all major conjugates, indicating interconversion of conjugates and breakdown to free 1-NAA. A regulatory role of conjugation in maintaining a particular level of free 1-NAA in the tissue is proposed herein.     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Polar transport of 1-naphthaleneacetic Acid determines the distribution of flower buds on explants of tobacco

Upon addition of 1-naphthaleneacetic acid (1-NAA) and benzylaminopurine, flower buds developed on explants from flower stalks of Nicotiana tabacum L. cv Samsun cultured in vitro. At low concentrations of 1-NAA, buds emerged mainly at the basal edge, whereas at high concentrations they developed on the remaining surface. The optimum concentrations for the two groups of buds were 0.45 micromolar and 2.2 micromolar, respectively, and the shapes of the concentration versus response curves were similar. The level of benzylaminopurine in the medium affected neither the shape nor the optimum concentration of these curves. The distribution of the buds over the explants was shown to be caused by polar auxin transport, leading to accumulation at the basal side. First, in the presence of the inhibitors 2,3,5-triiodobenzoic acid and 1-naphthylphthalamic acid, both groups of buds had the same optimum concentration of 1 micromolar 1-NAA. Second, after 6 hours of culture applied 1-NAA had accumulated in the basal part of the explant. In the presence of 1-naphthylphthalamic acid, no transport or accumulation of applied 1-NAA occurred.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Role of IAA conjugates in inducing ethylene production by tobacco leaf discs

Indole-3-acetic acid (IAA) labeled in its carboxyl group was metabolized by tobacco leaf discs (Nicotiana tabacum L. cv. Xanthi) into three metabolites, two of which were preliminarily characterized as a peptide and an ester-conjugated IAA. Reapplication of each of the three metabolites (at 10 μM) resulted in a marked stimulation of ethylene production and decarboxylation by the leaf discs. Similarly, these three IAA metab olites could induce elongation of wheat coleoptile segments, which was accompanied by decarboxylation. Both the exogenously supplied esteric and peptidic IAA conjugates were converted by the leaf discs into the same metabolites as free IAA. (1-¹⁴C)IAA, applied to an isolated epidermis tissue, was completely metabolized to the esteric and peptidic IAA conjugates. This epidermis tissue showed much higher ethylene production rates and lower decarboxylation rates than did the whole leaf disc. The results suggest that the participation of IAA conjugates in the regulation of various physiological processes depends on the release of free IAA, which is obtained by enzymatic hydrolysis of the conjugates in the tissue. The present study demonstrates biological activity of endogenous IAA conjugates that were synthesized by tobacco leaf discs in response to exogenously supplied IAA.     

Effect of Photoperiodic Floral Induction on the Metabolism of a Gibberellin Precursor, (-)-Kaurene, in Pharbitis nil

Pharbitis nil seedlings rapidly metabolized (-)-kaurene-17-¹⁴C administered to the cotyledons. Less than 20% of the radioactivity was recovered by extraction of the cotyledons on the following day. Of this the major metabolite was an unidentified acidic material which did not correspond chromatographically to any of the known gibberellins.     

Correlation between Polyamines and Pyrrolidine Alkaloids in Developing Tobacco Callus

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) α-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) α-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.     

Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates,free ecdysteroids,and ecdysteroid acids

[¹⁴C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.     

Polyamines, hydroxycinnamoylputrescines, and root formation in leaf explants of tobacco cultivated in vitro: effects of the suicide inhibitors of putrescine synthesis

In vitro formation of roots is obtained directly, without intermediate growth of callus, from foliar explants of a tobacco (Nicotiana tabacum) plant cultured on Murashige and Skoog medium containing IAA. Auxin-induced root formation was accompanied by significant changes in hydroxycinnamoylputrescine levels. Increasing levels were found in leaf explants during the first 14 days in culture; this was followed by a sharp decline after 20 days. Early changes in putrescine conjugates were detected in leaf explants before the visible appearance of roots. An early and transitory accumulation of hydroxycinnamoylputrescines was observed in the roots. Free polyamines (putrescine, spermidine, and spermine) in leaf explants and roots were always at a low level and only small changes in their concentrations were observed, α-dl-difluoromethylarginine and α-dl-difluoromethylornithine, specific, irreversible inhibitors of arginine decarboxylase and ornithine decarboxylase, respectively, inhibited putrescine accumulation and root initiation and reduced the fresh and dry weights of leaf explants. These effects were reversed by free putrescine or hydroxycinnamoylputrescines. The results reported here suggest that hydroxycinnamoylputrescines are associated with root formation. The relationship among free polyamines, hydroxycinnamoylputrescines, cell division, and root formation is discussed.     

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis

The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.4 micromolar BA showed that leaf explants from petunia line St40 required a shoot induction period of 6 to 10 days for commitment to shoot organogenesis; whereas leaf explants from petunia TLV1 required 12 to 28 days. The short induction period of petunia St40 and the higher organogenic response was positively associated with a threefold higher absorption of BA from the medium, an increased BA ribotide metabolite pool, the presence of BA within the explant during the shoot induction period, and the production of an unidentified metabolite C. However, the study of petunia TLV1 leaf explants showed that neither BA nor metabolite C are required during the shoot induction period for eventual shoot development. The longer shoot induction period of TLV1 was associated with low BA uptake during 24 days, a decreasing ribotide metabolite pool, the absence of benzyl adenosine triphosphate and metabolite C throughout the study, and the absence of BA within the explant during the shoot induction period. Differences in the shoot organogenic response of these related plant lines have been shown to be associated with differences in exogenous cytokinin uptake and the subsequent metabolism of that hormone.     

Folylpolyglutamates in Leishmania major

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.     

Metabolism of Auxin in Tomato Fruit Tissue: Formation of High Molecular Weight Conjugates of Oxindole-3-Acetic Acid via the Oxidation of Indole-3-Acetylaspartic Acid

High performance liquid chromatography of extracts of tomato (Lycopersicon esculentum Mill.) incubated with a relatively low concentration (4 μm) of [1-¹⁴C]indole-3-acetic acid (IAA) revealed the presence of two major polar metabolites. Hydrolysis of the two metabolites with 7 n NaOH yielded the same compound, which had a retention time similar to that of ring-expanded oxindole-3-acetic acid (OxIAA) on high performance liquid chromatography. The identity of the indolic moiety of these conjugates as OxIAA was further confirmed by gas chromatography-mass spectrometry. Chromatography of the two OxIAA conjugates on a calibrated Bio-Gel P-2 column indicated that their molecular weights are about 1200 and 1000. Aspartic acid and glutamic acid were the major amino acids detected in acid hydrolysates of the two conjugates. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of indole-3-acetylaspartic acid (IAAsp) with a concomitant decrease in the formation of the two OxIAA conjugates. Feeding experiments with labeled IAAsp and OxIAA showed that IAAsp and not OxIAA is the precursor of these conjugates. The data obtained indicate that exogenous IAA is converted in tomato pericarp tissue to high molecular weight conjugates, presumably peptides, of OxIAA via the oxidation of IAAsp. The oxidation of IAAsp seems to be a rate-limiting step in the formation of these conjugates from exogenous IAA.     

Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D

An auxin-requiring soybean root callus metabolized [1-¹⁴C]-2,4-dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-¹⁴C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites.     

The effect of light on metabolism of IAA in maize seedlings

Carbon 14-labelled indole-3-acetic acid (IAA) was fed to segments of shoots of Zea mays seedlings grown in light or dark to find the effect of light on IAA metabolism. The seedling parts coleoptile, with enclosed leaf, and mesocotyl were also used to examine differences in IAA metabolism between tissue types. The rate of metabolite formation as a function of time ranging from 1 to 12 hours was determined. Light did not significantly influence the amount of IAA taken up, but significantly increased its rate of metabolism and greatly increased the content of amide conjugates formed. There were also differences in metabolism depending on tissue type. In all tissues, IAA was metabolized mainly into six compounds. Four were tentatively identified as IAA-glucose (IAGlc), IAA-myo-inositol} (IAInos), indole acetamide (IAAm) and IAA-aspartic acid (IAAsp). 1-O-IAA-D-glucose (1-O-IAGlc) was the first conjugate formed and, except for mesocotyls in the light, it was the most abundant conjugate in maize tissue. In mesocotyl tissue the conversion of IAA into IAAsp was greatly stimulated by light, and the biosynthesis of IAAsp exceeded that of IAGlc. Since light strongly inhibited the growth of the mesocotyl, it is possible that the stimulation of IAAsp synthesis by light causes depletion of free IAA with resultant inhibition of mesocotyl growth.     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

Biological activities of indoleacetylamino acids and their use as auxins in tissue culture

THE AUXIN ACTIVITIES OF A NUMBER OF INDOLEACETYLAMINO ACID CONJUGATES HAVE BEEN DETERMINED IN THREE TEST SYSTEMS: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-l-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-l-alanine and indoleacetylglycine. The other conjugates inhibit shoot formation weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-l-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.     

IAA Amino Acid Conjugates Induce Differentiation of Tracheary Strands in Lettuce Pith Explants

Each of four amino acid conjugates of IAA was able to replacethe IAA requirement for xylogenesis in lettuce pith explants,when supplied at concentrations ten to 100 times those optimalfor IAA. Tracheary development induced by these conjugates tendedto be slightly slower and less in amount than with IAA, andthe tracheary strands shorter and less regular. Responses differedsomewhat among the four conjugates: IAA-D, L-aspartate gavedevelopment most like that with free IAA, and IAA-D, L-phenylalanineoften yielded the weakest tracheary development, while responsesto IAA-L-alanine and IAA-glycine were intermediate. The resultsare interpreted in terms of the ‘bound’ IAA conjugatesdiffusing into the pith explants and becoming xylogenic onlyon hydrolysis to ‘free’ IAA. As tracheary strandformation is believed to result from IAA fluxes, it seems thatthe free IAA also moved through the discs, presumably towardsthe surfaces where it degrades rapidly. Tracheary strand formationin these explants can be compared with vascular strand formationin the normal shoot tip, where IAA conjugates (auxin ‘precursors’)move acropetally and are hydrolysed to free IAA especially inthe young leaf primordia, we suggest, yielding local sourcesof IAA which may contribute both to the phyllotactic spacingof primordia and, moving basipetally, to the definition of theauxin pathways that develop as procambial strands behind individualleaf primordia. Lactuca sativa, lettuce, IAA conjugates, tracheary element differentiation, pith explants, xylem strands     

The metabolism of shikimate in the rat.

In the rat, shikimate was metabolized and excreted as hippurate, hexahydrohippurate, 3,4,5,6-tetrahydrohippurate, t-3,t-4-dihydroxycyclohexane-r-1-carboxylate and c-3,t-4-dihydroxycyclophexane-r-1-carboxylate, conjugates of catechol and CO2. The metabolism was entirely dependent on various initial microbial transformations in the gut, metabolite formation being suppressed in animals pretreated with antibiotics. Shikimate was not metabolized by mammalian tissues, and products of microbial metabolism were excreted either unchanged or after further biotransformation in the animal tissues.     

Identification of an unusual cyclooxygenase metabolite of arachidonic acid in rabbit renal medulla

A renal medulla 100,000g pellet metabolized arachidonic acid, C_20:4, to the previously described prostaglandins prostaglandin E₂, 6-ketoprostaglandin F_1α, thromboxane B₂, 12-hydroxyheptadecatrienoic acid, and 11-hydroxyeicosatetraenoic acid. In addition, under conditions of low enzyme to substrate ratios, the renal medulla also produced an unusual metabolite from arachidonic acid. This metabolite was inhibited by indomethacin, and thus suggested that it was a product of the cyclooxygenase. Addition of GSH to the incubation inhibited its formation, while p-hydroxymercuri-benzoate enhanced its formation. This compound was identified by HPLC purification, uv absorption, and gas chromatography-mass spectroscopy. The compound was 9,15 dioxo,11-hydroxyprosta-5,13-dienoic acid.     

Feruloylputrescine and Caffeoylputrescine Are Not Involved in Growth and Floral Bud Formation of Stem Explants from Nicotiana tabacum L. var Xanthi nc

The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). dl-α-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while dl-α-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media.