Biosynthesis and Metabolism of Indol-3yl-acetic acid: II. IN VIVO EXPERIMENTS WITH 14C-LABELLED PRECURSORS OF IAA IN TOMATO AND BARLEY SHOOTS

Our previous investigation of the naturally occurring indolecompounds in barley and tomato shoots suggested that the biosynthesisof indol-3yl-acetic acid (IAA) from tryptophan might proceedvia either the indol-3yl-pyruvic acid or tryptamine pathwaysin both species. The results further indicated that the indol-3yl-lacticacid pathway for IAA formation might also be operative in tomato.In the present study, tryptophan-3-¹⁴C and tryptamine-2-¹⁴Cwere fed to excised shoots of both barley and tomato, and indol-3yl-lacticacid-3-¹⁴C was also fed to shoots of tomato. All three compoundswere found to give rise to radioactive IAA with little dilutionin specific activity. Feeding tryptophan-3-¹⁴C also resultedin the labelling of indol-3yl-pyruvic acid, indol-3yl-acetaldehyde,and tryptamine, which were isolated and chemically identifiedfrom both species, and radioactive indol-3yl-lactic acid andtryptophol were also produced in tomato. Indol-3yl-acetaldehydewas found to be labelled in both species after administrationof tryptamine-2-¹⁴C, while the principal metabolite of indol-3yl-lacticacid-3-¹⁴C was radioactive tryptophan. These findings, alongwith the results from a quantitative study of the radioactivemetabolites, indicate that both the indol-3yl-pyruvic acid andtryptamine pathways can operate in both species, while the formationof IAA from indol-3yl-lactic acid in tomato probably occursindirectly, via tryptophan. These conclusions were supportedby the demonstration of the enzymes, L-tryptophan transaminase,L-trypto-phan decarboxylase, and indol-3yl-acetaldehyde dehydrogenasein cell-free extracts of both tissues, and of indol-3yl-pyruvicacid decarboxylase in the tomato extract. No indol-3yl-lacticacid decarboxylase activity was observed in the extracts fromeither tissue.     

Indole-3-acetic acid and 2-(indol-3-ylmethyl)indol-3-yl acetic acid in the thermophilic archaebacterium Sulfolobus acidocaldarius.

Indole-3-acetic acid (IAA) and 2-(indol-3-ylmethyl)indol-3-yl acetic acid were identified in lipid extracts of Sulfolobus acidocaldarius; they occurred at concentrations of 0.57 and 0.59 mumol/g (dry weight), respectively. The amount of IAA found in these cells is more than a thousand times greater than that found in a typical extract of a plant in which IAA serves as a plant growth hormone. Neither of these compounds was detected in the other archaebacteria that were analyzed; these included Sulfolobus sulfataricus, Halobacterium salinarium, and several strains of methanogenic bacteria. This is the first report of the natural occurrence of 2-(indol-3-ylmethyl)indol-3-yl acetic acid.     

The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem

Correlatively inhibited pea shoots (Pisum sativum L.) did not transport apically applied ¹⁴C-labelled indol-3yl-acetic acid ([¹⁴C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2° C) after decapitation. Rates of net uptake of [¹⁴C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-1-naphthylphthalamic acid. Uptake of the pH probe [¹⁴C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [¹⁴C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA efflux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid This work was supported by grant no. GR/D/08760 from the U.K. Science and Engineering Research Council. We thank Mrs. R.P. Bell for technical assistance.     

Auxin Physiology of Decapitated Stems of Phaseolus vulgaris L. Treated with Indol-3yl-acetic Acid

Physiological amounts of indol-3yl-acetic acid (IAA) were accumulatedby decapitated stems of Phaseolus vulgaris L. seedlings fromlanolin pastes, containing 0.1 per cent IAA, applied to thecut surfaces of the stumps. Both the levels and gradients ofextractable and diffusable IAA detected in the treated stumpscompared favourably with those reported for the whole plants.A considerable portion of IAA that entered the tissue was metabolizedto a compound that had chromatographic properties similar toindol-3yl-acetylaspartic acid. Two other metabolities were tentativelyidentified as indol-3yl-acetylaspartic acid indol-3yl-acetylglucose.The accumulated IAA appears to be transported as indol-3yl-aceticacid at an apparent velocity of 28 mm/h down the decapitatedinternodes.     

Shoots from MM. 106 apple rootstocks grown in a polythene tunnel do not contain a higher IAA concentration than shoots from field-grown plants

MM. 106 apple rootstock plants grown in a polythene tunnel show greater apical dominance and a higher propensity to root as cuttings than plants grown in the field. Experiments were conducted to test the hypothesis that the growth habit and rooting behaviour of polythene tunnel plants were caused by increased concentrations of idole-3yl-acetic acid. Cuttings taken from field-grown plants which had been sprayed with IAA showed increased rooting. In shoots of both field-grown and polythene tunnel-grown plants endogenous IAA levels were highest in the upper shoot region and declined progressively with distance from the apex. Plants grown in the polythene tunnel, however, did not contain significantly higher IAA levels than field plants. The analytical data do not support the hypothesis that the growth and rooting behaviour of plants grown in a polythene tunnel were caused by increased concentrations of IAA.Abbreviations IAA indol-3yl-acetic acid     

The influence of small direct electric currents on the transport of auxin in intact plants

When a d.c. potential of 9.0 V was applied to the stem of intact pea seedlings (Pisum sativum L. cv. Meteor and cv. Alderman) via 10 mM KCl-soaked filter paper electrodes placed ca. 50 mm apart the stem passed a steady current of 15–20 A (resistance ca. 100 k cm^-1). The basipetal transport of [1-¹⁴C]IAA applied to the apical bud was completely inhibited over the portion of the stem through which current flowed and ¹⁴C-labelled compounds accumulated in the vicinity of the upper electrode. The inhibition of transport was independent of the polarity of the applied potential. The basipetal transport of IAA in the stem above the electrode was not affected.Labelled auxin accumulated at the upper electrode both as unchanged IAA and as a compound tentatively identified as indol-3yl-acetyl aspartic acid (IAAsp). These compounds were only slowly remobilised when the current was interrupted. However, the ability of the transport system to move freshly-applied IAA was rapidly and fully restored when the potential was removed. No injury to the plant was detected after maintaining a current flow for up to 72 h. No leakage of ¹⁴C-labelled compounds into the KCl solution bathing the electrodes was detected.Abbreviations IAA indol-3yl-acetic acid - IAAsp indol-3yl-acetyl aspartic acid     

Regulation of root formation by auxin-ethylene interaction in pea stem cuttings

The possibility was investigated that the inhibition of rooting in pea ( Pisum sativum L. cv. Weibull's Marma) cuttings caused by low indol-3yl-acetic acid (IAA) concentrations is due to ethylene produced as a result of IAA treatment. Treatment with 10 uμ IAA reduced the number of roots to about 50% of the control and increased ethylene production in the stem bases by about 20 times the control value during the two first days of treatment. Ethylene-releasing compounds (ethephon and 1-amino-cyclopropane-1-carboxylic acid, ACC), in concentrations giving a similar ethylene release, inhibited rooting to the same extent or more strongly than IAA. These results indicate that IAA-induced ethylene is at least responsible for the negative component in IAA action on root formation in pea cuttings. A higher IAA concentration (100 μ) and indol-3yl-butyric acid efficiently counteracted the negative effect of ethylene on root formation.     

Effect of Applied and Endogenous Auxin on Callus and Root Formation of In Vitro Shoots of the Apple: Rootstocks M26 and A2

The influence of exogenous IBA (indol-3yl-butyric acid) on rootand callus formation was studied in shoots of the apple rootstocksA2 and M26. The shoots grown in vitro were derived originallyfrom meristems of both juvenile and adult trees. Endogenousindol-3yl-acetic acid (IAA) concentrations in leaves and stemswere correlated with the responses to applied IAA. After 30 subcultures shoots from A2 and M26 rooted easily, butA2 did so more readily and even without IBA. Treatment withIBA improved percentage rooting and number of roots in bothrootstocks. Ex-adult and ex-juvenile shoots of A2 formed rootsto the same extent. However, ex-adult shoots of A2 showed ahigher IBA optimum for root number than ex-juvenile A2 and werealso less sensitive to supra-optimal IBA concentrations. Incontrast, in M26, there were no differences between ex-adultand ex-juvenile shoots. The results imply that rooting ability is associated more withdifferences between cultivars than with the origin of the explants.The best rooting occurred in ex-adult shoots of A2 which hadthe lowest endogenous IAA concentration, while callus formationwas correlated with high endogenous auxin concentration. Ex-adultA2 produced almost no callus even after exposure to high IBAconcentrations (25µM) whereas ex-adult M26 formed muchmore callus at 1/10 of the IBA concentration. Malus sylvestris (L.) Mill. var. domestica Borkh., Malus pumila Mill., apple rootstocks A2 and M26, in vitro culture, root and callus formation, HPLC analyses of IAA     

Auxin gradient along the root of the maize seedling

[5-³H]Indol-3yl-acetic acid (IAA) applied to the shoot apices of intact 6-day-old maize (Zea mays L.) plants moved into the primary root and accumulated at the root apex. IAA from the shoot could partially satisfy the requirement of the primary root for IAA for growth.Abbreviation IAA indol-3yl-acetic acid     

Endogenous and exogenous auxin in the control of root growth

The endogenous indol-3yl-acetic acid (IAA) of detipped apical segments from roots of maize (cv ORLA) was greatly reduced by an exodiffusion technique which depended upon the preferential acropetal transport of the phytohormone into buffered agar. When IAA was applied to the basal cut ends of freshly prepared root segments only growth inhibitions were demonstrable but after the endogenous auxin concentration had been reduced by the exodiffusion technique it became possible to stimulate growth by IAA application. The implications of the interaction between exogenous and endogenous IAA in the control of root segment growth are discussed with special reference to the role of endogenous IAA in the regulation of root growth and geotropism.Abbreviations IAA indol-3yl-acetic acid - GC-MS gas chromatography-mass spectrometry     

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-¹⁴C]-or [5-³H]IAA occurred at velocities (approx. 8–15 mm·h^-1) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-¹⁴C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-¹⁴C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m^-3). Transport velocity increased with concentration up to 83 mmol·m^-3 IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-¹⁴C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m^-3 NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-¹⁴C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m^-3 NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.Abbreviations IAA indol-3yl-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Auxin transport and the regulation of petiole abscission in cotton (Gossypium hirsutum L.): A comparison of indol-3yl-acetic acid and phenylacetic acid

Distal applications of indol-3yl-acetic acid (IAA) to debladed cotyledonary petioles of cotton (Gossypium hirsutum L.) seedlings greatly delayed petiole abscission, but similar applications of phenylacetic acid (PAA) slightly accelerated abscission compared with untreated controls. Both compounds prevented abscission for at least 91 h when applied directly to the abscission zone at the base of the petiole. The contrasting effects of distal IAA and PAA on abscission were correlated with their polar transport behaviour-[1-¹⁴C]IAA underwent typical polar (basipetal) transport through isolated 30 mm petiole segments, but only a weak diffusive movement of [1-¹⁴C]PAA occurred.Removal of the shoot tip substantially delayed abscission of subtending debladed cotyledonary petioles. The promotive effect of the shoot tip on petiole abscission could be replaced in decapitated shoots by applications of either IAA or PAA to the cut surface of the stem. Following the application of [1-¹⁴C]IAA or [1-¹⁴C]PAA to the cut surface of decapitated shoots, only IAA was transported basipetally through the stem. Proximal applications of either compound stimulated the acropetal transport of [¹⁴C]sucrose applied to a subtending intact cotyledonary leaf and caused label to accumulate at the shoot tip. However, PAA was considerably less active than IAA in this response.It is concluded that whilst the inhibition of petiole abscission by distal auxin is mediated by effects of auxin in cells of the abscission zone itself, the promotion of abscission by the shoot tip (or by proximal exogenous auxin) is a remote effect which does not require basipetal auxin transport to the abscission zone. Possible mechanisms to explain this indirect effect of proximal auxin on abscission are discussed.     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

A tracheid-differentiation factor from pine needles

Exogenous indol-3yl-acetic acid (IAA), alone and together with several cytokinins, was ineffective in promoting the complete differentiation into tracheids of cambial derivatives of Pinus contorta Dougl.; IAA alone promoted cambial cell division and primary-wall growth in cambial derivatives throughout the stem's length. In contrast, a single pair of needles on a stem cutting in light promoted neither cambial cell division nor primary-wall growth in cambial derivatives but did promote complete differentiation of cambial cells into tracheids; tracheids differentiated only near the junction of the foliated short shoot with the stem. Clear inter-and intracellular differences in the extent of tracheid differentiation occurred in response to a single needle pair and have suggested the hypothesis that a specific tracheid-differentiation factor regulates the differentiation of cells into proto-, meta-, or secondary-xylem tracheary elements through an interaction with IAA.Abbreviations IAA indol-3yl acetic acid - K kinetin - BAP 6-benzylaminopurine - Z zeatin - ZR zeatin riboside - FAA formalin: acetic acid: ethanol: water (10:5:50:35, by vol.)     

Early Responses of Excised Stem Segments to Auxins

The elongation rate of lupin hypocotyl and pea stem segmentswas measured every minute after the addition of various auxinsand auxin precursors. After a latent period the growth-rateincreased to a peak, fell to a minimum, and with most compoundsincreased to a second maximum. Compounds used include indol-3yl-aceticacid (IAA), indol-3yl-acetamide, naphth-2yl-oxyacetic acid,naphth- lyl-acetamide, and indol-3yl butyric acid. The extensibilityof the cell walls of the lupin segments was measured with anInstron Universal Testing Instrument at intervals after theaddition of IAA and it was shown that the lag period beforethe extensibility increased was longer than that for the growth-rate. Kinetic studies were made of the effect of Actinomycin D onIAA-induced growth. RNA synthesis during the first 20 and 40min after IAA addition was also examined in segments exposedto labelled RNA precursors during these times. The results supportthe conclusion that RNA synthesis is not necessary for the initialaction of auxin on elongation.     

A Novel Metabolic Pathway for Indole-3-Acetic Acid in Apical Shoots of Populus tremula (L.) x Populus tremuloides (Michx.)

Metabolism of indole-3-acetic acid (IAA) in apical shoots of Populus tremula (L.) x Populus tremuloides (Michx.) was investigated by feeding a mixture of [12C]IAA, [13C6]IAA, and [1[prime]-14C]IAA through the base of the excised stem. HPLC of methanolic plant extracts revealed eight major radiolabeled metabolites after a 24-h incubation period. Comparison between feeds with [5-3H]IAA and [1[prime]-14C]IAA showed that all detectable metabolites were nondecarboxylative products. The purified radiolabeled HPLC fractions were screened by frit-fast atom bombardment liquid chromatography-mass spectrometry for compounds with characteristic fragment pairs originating from the application with 12C and 13C isotopes. Samples of interest were further characterized by gas chromatography-mass spectrometry. Using this procedure, oxindole-3-acetic acid (OxIAA), indole-3-acetyl-N-aspartic acid (IAAsp), oxindole-3-acetyl-N-aspartic acid (OxIAAsp), and ring-hydroxylated oxindole-3-acetic acid were all identified as IAA metabolites. Furthermore, a novel metabolic pathway from IAA via IAAsp and OxIAAsp to OxIAA was established on the basis of refeeding experiments with the different IAA metabolites.     

Changes in the Levels of Endogenous Growth Regulators during Development of the Flowers of Chrysanthemum morifolium

Gibberellin-like substances and an auxin similar to IAA weredetected by bioassays in extracts of flowers of Chrysanthemummorifolium. The activity of these substances was shown to reacha maximum early in the development of the flower when its relativegrowth-rate was at a maximum, and then to decline with the relativegrowth-rate. The leaves of lateral flowering shoots were found to containgibberellins similar to those detected in the flowers whilea different gibberellin, which appeared to decrease in activitywith the age of the shoot, was detected in the stem. An auxinsimilar to indol-3yl-acetic acid (IAA) was also detected inthese stems. Growth-promoting substances were not detected inthe old stems and leaves from the main shoot. Gas-liquid chromatographyrevealed the presence of a number of additional gibberellinsin the flowers. The chemical nature of the growth substances is discussed inrelation to their biological and chromatographic behaviour.     

The Relationship between Flower Abortion and Endogenous Auxin Content of Rose Shoots

The amount of endogenous growth substances in stem, flowers and leaves of rose plants grown under different temperature and light conditions has been determined. It appeared to be two main growth promoting factors in the acidic fraction of the ether extract. One of them is assumed to be an auxin, probably indol-3yl-acetic acid (IAA); the other is not identified. The level of auxin was much higher in extracts from shoots grown at high temperature than in shoots grown at low temperature. Increasing light intensity also seemed to increase the auxin content of the shoots. Shoots which developed after a high cut back of the rose stem had a higher auxin content than shoots which developed after a low cut back. These findings are discussed in relation to the effect of temperature, light intensity and cut back practise on blind shoot formation in roses. The result of these investigations strongly indicate that abortion in roses is promoted by a low auxin level in the shoots.     

Adventitious root formation 'in vitro' in apple rootstocks (Malus pumila) I. Factors affecting the length of the auxin-sensitive phase in M.9

The length of the auxin-sensitive phase of root initiation 'in vitro' in the apple rootstock M.9 ( Malus pumila Mill.) has been determined using the auxins indol-3yl-acettc acid (IAA) at 2.8 × 10⁻⁵ M and indol-3yl-butyric acid (IBA) at 1.5 × 10⁻⁵ M in the presence and absence of 10⁻³ M phloroglucinol (PG). PG synergised IBA-induced rooting after 4 days exposure, but contact times exceeding 8 days decreased root number. In contrast, PG consistently synergised IAA-induced rooting in the dark for contact periods up to 13 days with the highest rooting being recorded at 9 days. An irradiance of 20 W m⁻² from fluorescent lamps halved IAA-induced rooting irrespective of the presence or absence of PG. The culture of shoots at temperatures of 22,25 and 29°C during the root initiation phase (auxin present) and the root emergence phase (auxin absent) produced no difference in rooting response. In the presence of PG the use of liquid culture in place of agar-solidified culture during the auxin-sensitive phase reduced root number but not rooting percentage.