Polar transport and content of indole-3-acetic acid in wounded sweet potato root tissues

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable in the proximalside and developed in the acropetal direction. We assumed thatthe polarity resulted from the increase in polar movement ofindoleacetic acid (IAA) (1977, Plant Physiol. 60: 563–566).Transport of IAA and change of the IAA level in the woundedtissue of sweet potato roots were investigated. Transport ofthe label from ¹⁴C-IAA was obviously polarized in the acropetaldirection. ¹⁴C-IAA administered to the wounded tissue was mainlymetabolized into two conjugates of IAA. The amount of IAA inthe wounded tissue, determined by the spectrofluorometric method,increased about 3-fold after 18 hr of incubation prior to thedevelopment of activities of some enzymes. The increase in IAAcontent was not affected with aseptic incubation, therefore,the possibility of IAA production by microorganisms on the woundedtissue was excluded. The results obtained strongly support ourhypothesis that IAA plays an important role in the metabolicresponse to wounding. (Received May 2, 1979; )     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

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     

Identification of oxindole-3-acetic acid,and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds

Oxindole-3-acetic acid (OxIAA) has been identified in germinating seeds of Scots pine (Pinus sylvestris) using gas chromatography-mass spectrometry. Seeds germinated for 5 d contained 2.7 ng OxIAA·g^-1 (dry weight) whereas ungerminated seeds contained 0.2 ng·g^-1. Isotopically labelled OxIAA was formed in seeds incubated with [1-¹⁴C]-, [2-¹⁴C]- or [²H₅]indole-3-acetic acid.Abbreviations DDC sodium diethyldithiocarbamate - GC gas chromatography - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - MS mass spectrometry - OxIAA oxindole-3-acetic acid - PVP polyvinylpyrrolidone - TMS trimethylsilyl     

Channelling auxin action: modulation of ion transport by indole-3-acetic acid

The growth hormone auxin is a key regulator of plant cell division and elongation. Since plants lack muscles, processes involved in growth and movements rely on turgor formation, and thus on the transport of solutes and water. Modern electrophysiological techniques and molecular genetics have shed new light on the regulation of plant ion transporters in response to auxin. Guard cells, hypocotyls and coleoptiles have advanced to major model systems in studying auxin action. This review will therefore focus on the molecular mechanism by which auxin modulates ion transport and cell expansion in these model cell types.     

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out duringadventitious root formation in avocado microcuttings. Both auxinand conjugate were monitored in control cuttings (rooted inthe absence of auxin) as well as in cuttings treated with arooting promotor (IBA) or an auxin transport inhibitor (TIBA).Additionally, a histological study to follow root differentiationwas carried out. In control cuttings IAA levels remained constantthroughout the rooting process, however, in IB A-treated cuttingsIAA levels increased 2-fold during the first 6 d. Addition of200 µM TIBA induced a slight decrease of IAA levels andinhibited root formation. As for IAAsp levels, both control and IBA-treated cuttings showeda big increase before root differentiation occurred and as theprocess went on, a progressive decrease took place. However,in TIBA-treated cuttings IAAsp levels not only did not increasebut diminished progressively during the process. The role ofauxin conjugates during the rooting process of avocado is discussed. Key words: Avocado, IAA, IAAsp, rooting     

Transport of shoot- and cotyledon-applied indole-3-acetic acid to Vicia faba root

To clarify the participation of indole-3-acetic acid (IAA) originatingfrom the shoot in root growth regulation and the mechanism ofIAA translocation from shoot to root, the movement of ¹⁴C-IAAwhich was applied to the epicotyl or the cotyledon of Viciafaba seedlings was investigated. The radioactivity of IAA appliedto the cotyledon moved faster to the root tip than that appliedto the epicotyl. On the basis of the effect of 2,3,5-triiodobenzoic acid on IAAmovement, a comparison with ¹⁴C-glucose movement and autoradiographicexamination, the nature of IAA movement was concluded to bepolar transport from the epicotyl to the basal part of the roots,while IAA movement from the epicotyl to the cotyledon, fromthe basal part of roots to the apical part, and from the cotyledonto the epicotyl and to the root took place in the phloem. Theradioactivity from ¹⁴C-IAA applied to the cotyledon accumulatedin lateral root primordia and vascular bundles. These factssuggest that IAA produced in cotyledons may participate in theregulation of Vicia root development. (Received December 21, 1979; )     

Influence of indole-3-acetic acid on adventitious root primordia of brittle willow

Summary Removal of the stem apex and certain leaves and axillary buds of brittle willows (Salix fragilis) was employed to limit the supply of endogenous auxin to adventitious root primordia during their formation, which occurs at predetermined sites. Limiting endogenous auxin by this surgical treatment resulted in reduced primordium initiation and, to a lesser degree, primordium growth in cell number. Root primordium cells in surgically treated plants differentiated into mature parenchyma after losing their meristematic character. Application of indole-3-acetic acid (IAA) to surgically treated plants partially overcame the effects of the surgical tretament, increasing root primordium initiation and growth by cell division. When IAA-2-¹⁴C was applied to surgically treated plants, label was detected in root primordium cells by means of autoradiography. Root primordium cells took up more label during the earliest stage of initiation than during a later stage of growth. The data indicate that the initiation of these primordia is more dependent on a supply of auxin than is their subsequent development. Further, the auxin apparently acts directly in the cells which initiate primordia.This investigation was supported in part by Public Health Service Research Grant No. UI 00110-07 (now 5R01 FD 00074-09) from the National Center for Urban and Industrial Health. Paper No. 7138, Min nesota Agricultural Experiment Station.     

Identification of 4-chloroindole-3-acetic acid and indole-3-aldehyde in seeds of Pinus sylvestris

4-Chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-aldehyde (IAId) have been characterized as endogenous constituents in seeds of Pinus sylvestris L. by gas chromatography-mass spectrometry. Quantitative estimates indicate that immature seeds contained 640 pg 4-Cl-IAA (g fresh weight)^-1 while mature seeds contained 340 pg (g dry weight)^-1. 4-Cl-IAA could not be detected in seeds five days after germination. The content of IAld increased from 127 pg (g dry weight)^-1 in mature seeds to 315 pg (g dry weight)^-1 after five days of germination.     

Uptake and metabolism of indole-3-butyric acid and indole-3-acetic acid by Petunia cell suspension culture

The uptake and metabolism of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were studied in suspension cell cultures of Petunia hybrida. The initial uptake of ³H-IBA was much higher than that of ³H-IAA, and after 10 min of incubation with labeled IBA and IAA, 4.6 pM vs 0.35 (39% vs 12% of total applied radioactivity) respectively, were found in the cell extracts. The uptake of IBA reached a plateau of 6.0 pM (62%) after 2 h while that of IAA increased continuously up to 1.5 pM (46%) after 24 h. Following the addition of 40 µM of unlabeled auxin more IBA was taken in initially than IAA (39% vs 12%), but the level almost equalized after 24 h of incubation when IBA uptake reached 890 nM (55%) and IAA 840 nM (46%).IBA was metabolized very rapidly by Petunia cell suspension to new compounds. HPLC of the cell extracts demonstrated a new metabolite after only 2 min of incubation, and after 30 min 60% of the radioactivity was in the new metabolite vs 10% in the IBA. The new compound was resolved by autofluorography to two metabolites but after 24 h only one metabolite was present. The IBA metabolites were identified tentatively as IBA aspartic acid (IBAasp) and IBA glucose (IBAglu). In the medium IBA disappeared at a fast rate and after 24h most of the radioactivity was present in the new metabolite, probably IBAasp. IAA was also converted rapidly to two new metabolites and both were still present after 24 h. No attempt was made to identify the metabolites of IAA. IAA metabolism proceeded at a slower rate, and autofluorography showed that while free IBA disappeared after 0.5 h, free IAA was still present after 1 h of incubation. We postulate that Petunia cells conjugate IBA rapidly to IBAglu which in turn is converted to form IBAasp which probably acts as a slow release hormone. Only intact cells were able to metabolize IBA and the reaction was affected by low temperature and anaerobic conditions. The fast rate of IBA uptake, the need for whole cells for the metabolism to proceed, and the fast change of IBA to a new metabolite in the medium, all suggest that both uptake and metabolism of IBA in Petunia cells occur on the cell surface.     

Metabolism of indole-3-acetic acid in rice: identification and characterization of N-beta-D-glucopyranosyl indole-3-acetic acid and its conjugates

A search was made for conjugates of indole-3-acetic acid (IAA) in rice (Oryza sativa) using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in order to elucidate unknown metabolic pathways for IAA. N-beta-d-Glucopyranosyl indole-3-acetic acid (IAA-N-Glc) was found in an alkaline hydrolysate of rice extract. A quantitative analysis of 3-week-old rice demonstrated that the total amount of IAA-N-Glc was equal to that of IAA. A LC-ESI-MS/MS-based analysis established that the major part of IAA-N-Glc was present as bound forms with aspartate and glutamate. Their levels were in good agreement with the total amount of IAA-N-Glc during the vegetative growth of rice. Further detailed analysis showed that both conjugates highly accumulated in the root. The free form of IAA-N-Glc accounted for 60% of the total in seeds but could not be detected in the vegetative tissue. An incorporation study using deuterium-labeled compounds showed that the amino acid conjugates of IAA-N-Glc were biosynthesized from IAA-amino acids. IAA-N-Glc and/or its conjugates were also found in extracts of Arabidopsis, Lotus japonicus, and maize, suggesting that N-glucosylation of indole can be the common metabolic pathway of IAA in plants.     

Lateral root formation in rice (Oryza Sativa L.): differential effects of indole-3-acetic acid and indole-3-butyric acid

Comparative effects of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on lateral root (LR) formation were studied using 2-day-old seedlings of IR8 rice (Oryza sativa L.). Results showed that IBA at all concentrations (0.8–500 nmol/L) increased the number of LRs in the seminal root. However exogenous IAA, failed to increase the number of LRs. On the other hand, both IBA and IAA caused inhibition of seminal root elongation and promotion of LR elongation, but IAA can only reach to the same degree of that of IBA at a more than 20-fold concentration. Exogenous IBA had no effect on endogenous IAA content. We conclude from the results that IBA could act directly as a distinct auxin, promoting LR formation in rice, and that the signal transduction pathway for IBA is at least partially different from that for IAA.     

Destruction of indole-3-acetic acid during the aerobic oxidation of sulfite

Indole-3-acetic acid (IAA) was rapidly destroyed in the presence of Mn²⁺, oxygen and sulfite ion. The optimal pH for the reaction was between 5 and 6. The destruction was dependent on the aerobic oxidation of sulfite, but was not inhibited by superoxide dismutase. Tracer studies indicate that IAA was converted into at least 3 compounds. Decarboxylation of IAA was not involved in the destruction.     

Promotion of hypocotyl elongation in loblolly pine (Pinus taeda L.) by indole-3-acetic acid

Elongation of excised loblolly pine ( Pinus taeda L.) hypocotyls was promoted by indole-3-acetic acid and the fungal metabolite, fusicoccin. Gibberellic acid, kinetin, zeatin, or zeatin-riboside were either without effect or promoted elongation only slightly. The most auxin-responsive tissue was just below the cotyledonary node, and elongation was confined to sections excised from the upper 2 cm of the hypocotyl. Indole-3-acetic acid induced elongation rates in the hypocotyl sections equal to those of intact hypocotyls when the sections were excised from young seedlings. Elongation rates decreased in intact hypocotyls before the excised tissues lost responsiveness to the auxin. Hypocotyl elongation in loblolly pine is discussed in relation to hypocotyl elongation in angiosperm species.     

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.