Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.     

Auxin conjugates: their role for plant development and in the evolution of land plants

Auxin conjugates are thought to play important roles as storage forms for the active plant hormone indole-3-acetic acid (IAA). In its free form, IAA comprises only up to 25% of the total amount of IAA, depending on the tissue and the plant species studied. The major forms of IAA conjugate are low molecular weight ester or amide forms, but there is increasing evidence of the occurrence of peptides and proteins modified by IAA. Since the discovery of genes and enzymes involved in synthesis and hydrolysis of auxin conjugates, much knowledge has been gained on the biochemistry and function of these compounds, but there is still much to discover. For example, recent work has shown that some auxin conjugate hydrolases prefer conjugates with longer-chain auxins such as indole-3-propionic acid and indole-3-butyric acid as substrate. Also, the compartmentation of these reactions in the cell or in tissues has not been resolved in great detail. The function of auxin conjugates has been mainly elucidated by mutant analysis in genes for synthesis or hydrolysis and a possible function for conjugates inferred from these results. In the evolution of land plants auxin conjugates seem to be connected with the development of certain traits such as embryo, shoot, and vasculature. Most likely, the synthesis of auxin conjugates was developed first, since it has been already detected in moss, whereas sequences typical of auxin conjugate hydrolases were found according to database entries first in moss ferns. The implications for the regulation of auxin levels in different species will be discussed.     

Auxin metabolism in mosses and liverworts

The plant hormone auxin (indole-3-acetic acid, IAA) is involved in the control of many phenomena during plant development. By characterizing steady-state free and conjugated IAA levels using a stable isotope dilution method coupled with gas chromatography- selected ion monitoring- mass spectrometry, this paper provides a detailed characterization of IAA metabolism in five liverworts, four mosses, and two tracheophytes. Long-term IAA conjugation patterns were monitored by incubating actively growing tissue with (14)C-IAA and then analyzing the de novo synthesis of IAA conjugates with radioimaging techniques. The liverworts, mosses, and tracheophytes can be differentiated by the total amount of IAA metabolites, the proportion of free and conjugated IAA, the chemical nature of their IAA conjugates, and the rates of IAA conjugation. Our tentative conclusion is that the liverworts appear to employ a biosynthesis-degradation strategy for the regulation of free IAA levels, in contrast to the conjugation-hydrolysis strategy apparently used by the mosses and tracheophytes. Such alternative metabolic strategies may have profound implications for macroevolutionary processes in these plant groups.     

Evolutionary patterns in auxin action

This review represents the first effort ever to survey the entire literature on auxin (indole-3-acetic acid, IAA) action in all plants, with special emphasis on the green plant lineage, including charophytes (the green alga group closest to the land plants), bryophytes (the most basal land plants), pteridophytes (vascular non-seed plants), and seed plants. What emerges from this survey is the surprising perspective that the physiological mechanisms for regulating IAA levels and many IAA-mediated responses found in seed plants are also present in charophytes and bryophytes, at least in nascent forms. For example, the available evidence suggests that the apical regions of both charophytes and liverworts synthesize IAA via a tryptophan-independent pathway, with IAA levels being regulated via the balance between the rates of IAA biosynthesis and IAA degradation. The apical regions of all the other land plants utilize the same class of biosynthetic pathway, but they have the potential to utilize IAA conjugation and conjugate hydrolysis reactions to achieve more precise spatial and temporal control of IAA levels. The thallus tips of charophytes exhibit saturable IAA influx and efflux carriers, which are apparently not sensitive to polar IAA transport inhibitors. By contrast, two divisions of bryophyte gametophytes and moss sporophytes are reported to carry out polar IAA transport, but these groups exhibit differing sensitivities to those inhibitors. Although the IAA regulation of charophyte development has received almost no research attention, the bryophytes manifest a wide range of developmental responses, including tropisms, apical dominance, and rhizoid initiation, which are subject to IAA regulation that resembles the hormonal control over corresponding responses in seed plants. In pteridophytes, IAA regulates root initiation and vascular tissue differentiation in a manner also very similar to its effects on those processes in seed plants. Thus, it is concluded that the seed plants did not evolve de novo mechanisms for mediating IAA responses, but have rather modified pre-existing mechanisms already operating in the early land plants. Finally, this paper discusses the encouraging prospects for investigating the molecular evolution of auxin action.     

Auxin-conjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease

Auxin conjugates play a role in the regulation of free indole-3-acetic acid (IAA) content in plants. Not much is known about the enzymes involved in either conjugate synthesis or hydrolysis. In this study we have isolated and characterized an auxin conjugate hydrolase from Chinese cabbage seedlings and investigated it during the development of both the Chinese cabbage plants and the clubroot disease. The hydrolase isolated from light- and dark-grown seedlings accepted the amide conjugates indole-3-acetic acid-alanine (IAAla), IAA-phenylalanine (IAPhe), but not IAA-aspartate (IAAsp) as substrates. We also found a substantial amount of hydrolysis of an ester conjugate (IAA-glucose, IAGlu) in our enzyme preparation. The tentative reaction product IAA was identified by HPLC and subsequent GC-MS analysis. The pH optima for the different substrates were not identical, suggesting several hydrolase isoforms. After gel filtration chromatography we found at least two peaks containing different hydrolase isoforms. The isoform, which converted IAGlu to IAA, exhibited a molecular mass of ca 63 kDa, and an isoform of ca 21 kDa converted IAAla and IAPhe. The increased free IAA content in clubroot-diseased roots of Brassicaceae can be due to either de novo synthesis or release of IAA from conjugates. To answer this question free, ester- and amide-bound IAA was measured in 24- and 30-day-old leaves and roots of healthy and Plasmodiophora brassicae-infected Chinese cabbage, and the hydrolase activity with different substrates measured in the same tissues. The amide conjugates were dramatically enhanced in infected roots, whereas free IAA was only slightly enhanced compared to the control tissue. Hydrolase activity was also enhanced in clubbed roots, but the substrate specificity differed from that found in the seedlings. Especially, IAAsp hydrolysis was induced after inoculation with P. brassicae. We conclude that different auxin conjugates can be hydrolyzed at different developmental stages or under stress.     

Plant hormone conjugation: A signal decision

Tight regulation of the auxin hormone indole-3-acetic acid (IAA) is crucial for plant development. Newly discovered IAA antagonists are the amide-linked tryptophan conjugates of IAA and jasmonic acid (JA). JA-Trp and IAA-Trp interfered with root gravitropism in Arabidopsis, and inhibited several responses to exogenously supplied IAA. Relatively low concentrations of the inhibitors occurred in Arabidopsis, but Pisum sativum flowers contained over 300 pmole g⁻¹ FW of JA-Trp. DihydroJA was an even more effective inhibitor than JA-Trp, suggesting that Trp conjugates with other JA derivatives may also be functional. JA-Trp and IAA-Trp add to the list of documented bioactive amide hormone conjugates. The only other example is JA-Ile, the recently discovered jasmonate signal. These examples establish that conjugation not only inactivates hormones, but in some cases creates novel compounds that function in hormone signaling.Key words: jasmonic acid, indole-3-acetic acid, auxin, tryptophan, conjugate, plant hormone, signaling, amino acid, antagonistPlants hold an amazing capacity to auto-regulate their growth and respond to a host of environmental challenges. Since the early discovery of the first plant hormone, indole-3-acetic acid (IAA),¹ science has progressively unveiled ever more complex, and sometimes surprising, ways that plants manipulate hormones to optimize their growth and thwart their opponents. Until recently, the covalent coupling of hormones to sugars, amino acids and peptides was thought to be merely a way to dispose of excess hormone.² The amide linkage of IAA to Asp and Glu does indeed result in IAA catabolism, while IAA-Ala and IAA-Leu are inactive stored forms of IAA.³ But the perception that all hormone conjugates are inactive changed abruptly with the discovery that the isoleucine conjugate of jasmonic acid (JA-Ile) is an active hormonal signal.     

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.     

Auxins in the development of an arbuscular mycorrhizal symbiosis in maize

While the levels of free auxins in maize (Zea mays L.) roots during arbuscular mycorrhiza formation have been previously described in detail, conjugates of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with amino acids and sugars were neglected. In this study, we have therefore determined free, ester and amide bound auxins in roots of maize inoculated with Glomus intraradices during early stages of the colonization process. Ester conjugates of IAA and IBA were found only in low amounts and they did not increase in AM colonized roots. The Levels of IAA and IBA amide conjugates increased 20 and 30 days past inoculation (dpi). The formation of free and conjugated IBA but not IAA was systemically induced during AM colonization in leaves of maize plants. This implicated a role for auxin conjugate synthesis and hydrolysis during AM. We have therefore investigated the in vivo metabolism of 3H-labeled IBA by TLC but only slight differences between control and AM-inoculated roots were observed. The activity of auxin conjugate hydrolase activity measured with three different putative substrates showed a decrease in infected roots compared to controls. The fluorinated IBA analog TFIBA inhibited IBA formation in leaves after application to the root system, but was not transported from roots to shoots. AM hyphae were also not able to transport TFIBA. Our results indicate complex control mechanisms to regulate the levels of free and conjugated auxins, which are locally and systemically induced during early stages of the formation of an arbuscular mycorrhizal symbiosis.     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μ M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants ( gluS ) deficient in chloroplastic glutamate synthase.     

Induction And Characterization of an Indole-3-acetyl-L-alanine Hydrolase From Arthrobacter Ilicis

Indole-3-acetic acid (IAA)-amino acid amide conjugates have been found to be present in many plants, and they are proposed to function in the regulation of plant IAA metabolism in a variety of ways. IAA-amino acid conjugate hydrolase activities, and the genes that encode them, are therefore potentially important tools for modification of IAA metabolism, both for agronomic reasons as well as for determination of the mechanisms of IAA regulation. We have developed a simple and economical method to induce IAA-amino acid conjugate hydrolases in bacteria with N-acetyl-L-amino acids. Using this method, we identified four bacterial strains that can be induced to produce IAA-Ala hydrolases: Arthrobacter ureafaciens C-10, Arthrobacter ureafaciens C-50, Arthrobacter ilicis D-50, and Cellulomonas fimi D-100. The enzyme kinetics and the biochemical characteristics of IAA-Ala hydrolase from one specific bacterium, Arthrobacter ilicis D-50, have been determined. The enzyme has a unique substrate specificity for IAA-amino acid conjugates compared to a bacterial IAA-Asp hydrolase previously characterized.     

Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

The rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β-glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole-3-acetyl)-β-D-glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole-3-acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild-type and transgenic 35S-rolB tobacco plants and metabolic studies were carried out with [¹³C₆]IAA [2′-¹⁴C]IAA, [¹⁴C]IAGluc, [5-³H]-2-o-(indole-3-acetyl)-myo-inositol and [¹⁴C]indole-3-acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.     

Quantitation of indoleacetic Acid conjugates in bean seeds by direct tissue hydrolysis

Gas chromatography-selected ion monitoring-mass spectral analysis using [¹³C₆]indole-3-acetic acid (IAA) as an internal standard provides an effective means for quantitation of IAA liberated during direct strong basic hydrolysis of bean (Phaseolus vulgaris L.) seed powder, provided that extra precautions are undertaken to exclude oxygen from the reaction vial. Direct seed powder hydrolysis revealed that the major portion of amide IAA conjugates in bean seeds are not extractable by aqueous acetone, the solvent used commonly for IAA conjugate extraction from seeds and other plant tissues. Strong basic hydrolysis of plant tissue can be used to provide new information on IAA content.     

Characterization of a family of IAA-amino acid conjugate hydrolases from Arabidopsis

The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-l-amino acid conjugates to inhibit Arabidopsis seedling root growth. We have also determined the ability of purified glutathione S-transferase (GST) fusions of four Arabidopsis IAA-amino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates in vitro, and GST-ILR1, GST-IAR3, and GST-ILL2 have K(m) values that suggest physiological relevance. In vivo inhibition of root elongation correlates with in vitro hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.     

The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation

Indole-3-acetic acid (IAA) was oxidized by horseradish peroxidase, but ester and amide conjugates of IAA were not degraded. Addition of indoleacetyl-myo-inositol, indoleacetyl-L-aspartate, indoleacetylglycine, indoleacetyl-L-alanine, indoleacetyl-D-alanine, or indoleacetyl--alanine did not affect the rate of oxidation of IAA by horseradish peroxidase. Peroxidase preparations from Pisum sativum L. and Zea mays L. behaved similarly in that they rapidly oxidized IAA, but not conjugates found in the plant from which the peroxidase was prepared. These results indicate that conjugation could affect the stability of IAA in vivo.Abbreviation IAA Indole-3-acetic acid     

Indole-3-acetic acid and indole-3-acetylaspartic acid isolated from seeds of Heracleum laciniatum Horn

Seeds from mature flowers of Heracleum laciniatum were collected locally (Tromsø, Norway). Seed coats were removed and the seeds were analyzed for their content of free, free plus ester-conjugate, and total indole-3-acetic acid (IAA) by quantitative gas chromatography-mass spectrometry. Seeds contained high levels of free and amide-linked IAA relative to other dicotyledonous seeds for which values have been published. The major amide conjugate in this material was identified as indole-3-acetylaspartate by gas chromatography-mass spectrometry of its bis-methyl ester.     

Developmental regulation of indole-3-acetic acid turnover in Scots pine seedlings

Indole-3-acetic acid (IAA) homeostasis was investigated during seed germination and early seedling growth in Scots pine (Pinus sylvestris). IAA-ester conjugates were initially hydrolyzed in the seed to yield a peak of free IAA prior to initiation of root elongation. Developmental regulation of IAA synthesis was observed, with tryptophan-dependent synthesis being initiated around 4 d and tryptophan-independent synthesis occurring around 7 d after imbibition. Induction of catabolism to yield 2-oxindole-3-acetic acid and irreversible conjugation to indole-3-acetyl-N-aspartic acid was noticed at the same time as de novo synthesis was first detected. As a part of the homeostatic regulation IAA was further metabolized to two new conjugates: glucopyranosyl-1-N-indole-3-acetyl-N-aspartic acid and glucopyranosyl-1-N-indole-3-acetic acid. The initial supply of IAA thus originates from stored pools of IAA-ester conjugates, mainly localized in the embryo itself rather than in the general nutrient storage tissue, the megagametophyte. We have found that de novo synthesis is first induced when the stored pool of conjugated IAA is used up and additional hormone is needed for elongation growth. It is interesting that when de novo synthesis is induced, a distinct induction of catabolic events occurs, indicating that the seedling needs mechanisms to balance synthesis rates for the homeostatic regulation of the IAA pool.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

Effect of Exogenous Indole-3-Acetic Acid and Indole-3-Butyric Acid on Internal Levels of the Respective Auxins and Their Conjugation with Aspartic Acid during Adventitious Root Formation in Pea Cuttings

The influence of exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on the internal levels of these auxins was studied during the first 4 days of adventitious root formation in cuttings of Pisum sativum L. The quantitations were done by high performance liquid chromatography with spectrofluorometric detection. IBA, identified by combined gas chromatography-mass spectrometry (GC-MS), was found to naturally occur in this plant material. The root inducing ability of exogenous IBA was superior to that of IAA. The IAA level in the tissue increased considerably on the first day after application of IAA, but rapidly decreased again, returning to a level twice the control by day 3. The predominant metabolic route was conjugation with aspartic acid, as reflected by the increase in the level of indole-3-acetylaspartic acid. The IBA treatment resulted in increases in the levels of IBA, IAA, and indole-3-acetylaspartic acid. The IAA content rapidly returned to control levels, whereas the IBA level remained high throughout the experimental period. High amounts of indole-3-butyrylaspartic acid were found in the tissue after feeding with IBA. The identity of the conjugate was confirmed by ¹H-nuclear magnetic resonance and GC-MS. IBA was much more stable in solution than IAA. No IAA was detected after 48 hours, whereas 70% IBA was still recovered after this time. The relatively higher root inducing ability of IBA is ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. Adventitious root formation is discussed on the basis of these findings.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.