Quantitative Analysis of Indole-3-Acetic Acid Metabolites in Arabidopsis

A general gas chromatography/mass spectrometry (MS)-based screen was performed to identify catabolites and conjugates of indole-3-acetic acid (IAA) during vegetative growth of Arabidopsis. This experiment revealed the existence of two new conjugates: N-(indole-3-acetyl)-alfa-alanine (IA-Ala) and N-(indole-3-acetyl)-alfa-leucine (IA-Leu). A method for quantitative analysis of IAA metabolites in plant extracts by liquid chromatography-electrospray tandem MS has been developed. The accuracy and precision of the new method are better than 10% for standards close to the detection limit, and are between 6% and 16% for the entire protocol applied to plant extracts. The low detection limits, 0.02 to 0.1 pmol for the different metabolites, made it possible to use as little as 50 to 100 mg of tissue for quantitative analysis. The analysis was performed on different tissues of an Arabidopsis plant at two stages of development, using heavy labeled internal standards of the catabolite 2-oxoindole-3-acetic acid as well as IAA conjugated to amino acids: aspartate, glutamate, Ala, and Leu. Expanding leaves and roots that generally contain high amounts of the free hormone also contained the highest levels of IA-aspartate, IA-glutamate, and 2-oxoindole-3-acetic acid, supporting their role as irreversible catabolic products. The levels of IA-Leu and IA-Ala did not follow the general distribution of IAA. Interestingly, the level of IA-Leu was highest in roots and IA-Ala in the aerial tissues.     

A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues

A microscale technique has been developed for routine quantifications of picogram amounts of indole-3-acetic acid (IAA) in plant tissues by combined gas chromatography-mass spectrometry. Low- and high-resolution selected-ion-monitoring and selected-reaction-monitoring mass spectrometry techniques were compared for selectivity and precision. The best selectivity was obtained with selected-reaction-monitoring analysis, and 1-mg samples containing 500 fg of IAA could be analyzed accurately with this method. This technique was used to investigate the IAA distribution pattern along the longitudinal axis of tobacco (Nicotiana tabacum [L.]) leaves. In young, developing leaves an increase of endogenous IAA from the leaf tip to the base of the leaf was observed, whereas the level of IAA was uniform along this axis in mature leaves.     

Oxidation of Indole-3-Acetic Acid to Oxindole-3-Acetic Acid by an Enzyme Preparation from Zea mays

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.     

Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.     

Metabolism of Indole-3-Acetic Acid in Arabidopsis

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.The plant hormone IAA is an important signal molecule in the regulation of plant development. Its central role as a growth regulator makes it necessary for the plant to have mechanisms that strictly control its concentration. The hormone is believed to be active primarily as the free acid, and endogenous levels are controlled in vivo by processes such as synthesis, oxidation, and conjugation. IAA has been shown to form conjugates with sugars, amino acids, and small peptides. Conjugates are believed to be involved in IAA transport, in the storage of IAA for subsequent use, in the homeostatic control of the pool of the free hormone, and as a first step in the catabolic pathways (Cohen and Bandurski, 1978; Nowacki and Bandurski, 1980; Tuominen et al., 1994; Östin et al., 1995; Normanly, 1997). It is generally accepted that in some species conjugated IAA is the major source of free IAA during the initial stages of seed germination (Ueda and Bandurski, 1969; Sandberg et al., 1987; Bialek and Cohen, 1989), and there is also evidence that in some plants (but not all; see Bialek et al., 1992), the young seedling is entirely dependent on the release of free IAA from conjugated pools until the plant itself is capable of de novo synthesis (Epstein et al., 1980; Sandberg et al., 1987).The function of conjugated IAA during vegetative growth is somewhat less clear. It has been shown that conjugated IAA constitutes as much as 90% of the total IAA in the plant during vegetative growth (Normanly, 1997). However, the role of the IAA conjugates at this stage of the plant''s life cycle remains unknown. Analysis of endogenous IAA conjugates in vegetative tissues has revealed the presence of a variety of different compounds, including indole-3-acetyl-inositol, indole-3-acetyl-Ala, IAAsp, and IAGlu (Anderson and Sandberg, 1982; Cohen and Baldi, 1983; Chisnell, 1984; Cohen and Ernstsen, 1991; Östin et al., 1992). Studies of vegetative tissues have indicated that IAAsp, one of the major conjugates in many plants, is the first intermediate in an irreversible deactivation pathway (Tsurumi and Wada, 1986; Tuominen et al., 1994; Östin, 1995). Another mechanism that is believed to be involved in the homeostatic control of the IAA pool is catabolism by direct oxidation of IAA to OxIAA, which has been shown to occur in several plant species (Reinecke and Bandurski, 1983; Ernstsen et al., 1987).One area in the study of IAA metabolism in which our knowledge is increasing is the analysis of the homeostatic controls of IAA levels in plants. It has been possible, for instance, to increase the levels of IAA in transgenic plants expressing iaaM and iaaH genes from Agrobacterium tumefaciens. Analysis of these transgenic plants has indicated that plants have several pathways that can compensate for the increased production of IAA (Klee et al., 1987; Sitbon, 1992). It is expected that future studies using now-available genes will provide further insight into IAA metabolism. For example, a gene in maize encoding IAA-Glc synthetase has been identified, and several genes (including ILR1, which may be involved in hydrolysis of the indole-3-acetyl-Leu conjugate) have been cloned from Arabidopsis (Szerszen et al., 1994; Bartel and Fink, 1995). Furthermore, Chou et al. (1996) identified a gene that hydrolyzes the conjugate IAAsp to free IAA in the bacterium Enterobacter aggloremans.Because of its small genome size, rapid life cycle, and the ease of obtaining mutants, Arabidopsis is increasingly used as a genetic model system to investigate various aspects of plant growth and development. IAA signal transduction is also being investigated intensively in Arabidopsis in many laboratories (Leyser, 1997). Mutants with altered responses to externally added auxins or IAA conjugates have been identified in Arabidopsis. The identified mutants are either signal transduction mutants such as axr1-4 (Lincoln et al., 1990), or have mutations in genes involved in auxin uptake or transport, such as aux1 and pin1 (Okada et al., 1991; Bennett et al., 1996). A few mutants that are unable to regulate IAA levels or are unable to hydrolyze IAA conjugates, sur1-2 and ilr1, respectively, have also been identified (Bartel and Fink, 1995; Boerjan et al., 1995). To our knowledge, no mutant that is auxotrophic for IAA has been identified to date, which may reflect the redundancy in IAA biosynthetic pathways or the lethality of such mutants.In spite of the work reported thus far, many aspects of the metabolism of IAA in Arabidopsis require further investigation, because few details of the processes involved in IAA regulation are known. This lack of knowledge puts severe constraints on genetic analysis of IAA metabolism in Arabidopsis. For example, it is essential to have prior knowledge of IAA metabolism to devise novel and relevant screens with which to identify mutants of IAA metabolism. We have sought to address this issue by identifying the metabolic pathways involved in catabolism and conjugation under conditions that minimally perturb physiological processes. In this investigation we studied the conjugation and catabolic pattern of IAA by supplying relatively low levels of labeled IAA and identifying the catabolites and conjugates by MS. Different feeding systems were tested to optimize the application of IAA and to avoid irregularities in metabolism attributable to culturing, feeding conditions, or microbial activity. It is well documented that IAA metabolism is altered according to the amount of exogenous auxin applied; therefore, we placed special emphasis on distinguishing between catabolic routes that occur at near-physiological concentrations and those that occur at the high auxin concentrations commonly used in mutant screens.     

Metabolism of Indole-3-Acetic Acid: III. Identification of Metabolites Isolated from Crown Gall Callus Tissue

The metabolism of labeled indole-3-acetic acid (IAA-2-¹⁴C) was investigated in Parthenocissus tricuspidata crown gall callus tissue. After 48 hours incubation, 85 to 90% of the supplied IAA was taken up by the tissue, and of that taken up, about 45% was conjugated with five amino acids. The conjugates found were aspartic and glutamic acid (minor ones) as well as glycine, alanine, and valine (major ones). The last four are being reported for the first time as metabolites of IAA. These conjugates were identified through their chromatographic properties, hydrolysis products, and their mass spectra. The possible significance of these amino acid conjugates is discussed.     

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.     

新疆大花红景天化学成分的HPLC-MS研究

采用高效液相色谱-电喷雾-四极杆-飞行时间串联质谱法(HPLC-ESI-Q-TOF-MS),分别在正负离子模式下对新疆产大花红景天(Rhodiola crenulata)提取物进行分离分析。通过精确质量测定和同位素峰形匹配(Sig-maFit)确定了化合物的元素组成。在正离子模式下,选择[M+H]+或[M+NH4]+准分子离子进行二级裂解对其结构进行分析。根据物质的元素组成和二级质谱信息,初步确定了新疆大花红景天提取物中的12种化合物。     

Dioxindole-3-Acetic Acid Conjugates Formation from Indole-3-Acetylaspartic Acid in Vicia Seedlings

When indole-3-acetic acid (IAA) is applied to the cotyledonsof broad bean seedlings (Vicia faba L. cv Chukyo), the majormetabolites found in the roots are 3-(O-ß-glucosyl)-2-indoIone-3-acetylaspartic acid (Glc-DIA-Asp) and 3-hydroxy-2-indolone-3-acetylasparticacid (DIA-Asp). In this report, the metabolic pathway from IAAto the two dioxindole-3-acetic acid (DIA) conjugates was investigatedby using [¹⁴C]IAA, [¹⁴C]DIA, [¹⁴C]indole-3-acetylaspartic acid(IAA-Asp), and [¹⁴C]IAA-[³H]Asp. The precursor of DIA-Asp wasfound to be IAA-Asp but not DIA. Incorporation of the doublelabeled IAA-Asp into the DIA conjugates demonstrated that hydrolysisof IAA-Asp was not involved in the formation of the DIA conjugates.DIA-Asp was further metabolized to Glc-DIA-Asp in the cotyledons,while formation of Glc-DIA-Asp in the roots was very low. Glc-DIA-Aspformed in the cotyledons was transported to the roots. (Received April 21, 1986; Accepted September 10, 1986)     

Enhancement of Indole-3-Acetic Acid Photodegradation by Vitamin B6

Dear Editor, The plant hormone indole-3-acetic acid （IAA） has long been used in plant culture media for practical applications and sci- entific inquiries. The use of IAA is complicated by the fact that IAA is a photo-labile compound. In Murashige and Skoog （MS） plant media （Murashige and Skoog, 1962）, the concen- trations of salts and mineral nutrients are known to hasten the photodegradation of IAA under white light （Dunlap and Robacker, 1988）. This degradation can be virtually eliminated by the use of a yellow-colored light filter that removes UV, violet, and some of the blue wavelengths from the incident light （Stasinopoulos and Hangarter, 1990）. However, the use of yellow light clearly affects the quality of light that the plants under study receive. In addition to applications in plants, IAA has been used in human health applications.     

Immuno-Gold Localization of Indole-3-Acetic Acid in Peach Seedlings

The localization of indole-3-acetic acid (IAA) in peach seedlings(Prunus persica [L.] Batsch ‘Momo Daigi Tsukuba 4’)was investigated using immunocytochemical technique. In meristematiccells of root tip, the gold particles were accumulated in nucleolus,while in leaf cells, they were mainly associated to chloroplastsand mitochondria. Physiological meaning of these localizationswas discussed. (Received December 13, 1989; Accepted April 12, 1990)     

Indole-3-Acetic Acid (IAA) Oxidation by Leghemoglobin form Soybean

Leghemoglobin from nudules of soybean prepated by ammonium sulfate frationation appeared to be able to destroy substantial quantities of IAA, Degradation still occurred in purified leghemoglobin preparations isolated by G-100 Sephadex chromatography. Nicotinic acid and acetate affecting the conformation of leghemoglobin both inhibited IAA oxidation by this hemoprotein. The ferric form was found to be the most active in this catabolism. This oxication, related to the psedudoperocidase activity of leghemoghlobin, is undoubtedly restricted in vivo by the very low level of the ferric form present in efficient nodules.     

Conversion of Endogenous Indole-3-Butyric Acid to Indole-3-Acetic Acid Drives Cell Expansion in Arabidopsis Seedlings

Genetic evidence in Arabidopsis (Arabidopsis thaliana) suggests that the auxin precursor indole-3-butyric acid (IBA) is converted into active indole-3-acetic acid (IAA) by peroxisomal β-oxidation; however, direct evidence that Arabidopsis converts IBA to IAA is lacking, and the role of IBA-derived IAA is not well understood. In this work, we directly demonstrated that Arabidopsis seedlings convert IBA to IAA. Moreover, we found that several IBA-resistant, IAA-sensitive mutants were deficient in IBA-to-IAA conversion, including the indole-3-butyric acid response1 (ibr1) ibr3 ibr10 triple mutant, which is defective in three enzymes likely to be directly involved in peroxisomal IBA β-oxidation. In addition to IBA-to-IAA conversion defects, the ibr1 ibr3 ibr10 triple mutant displayed shorter root hairs and smaller cotyledons than wild type; these cell expansion defects are suggestive of low IAA levels in certain tissues. Consistent with this possibility, we could rescue the ibr1 ibr3 ibr10 short-root-hair phenotype with exogenous auxin. A triple mutant defective in hydrolysis of IAA-amino acid conjugates, a second class of IAA precursor, displayed reduced hypocotyl elongation but normal cotyledon size and only slightly reduced root hair lengths. Our data suggest that IBA β-oxidation and IAA-amino acid conjugate hydrolysis provide auxin for partially distinct developmental processes and that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.The auxin indole-3-acetic acid (IAA) controls both cell division and cell expansion and thereby orchestrates many developmental events and environmental responses. For example, auxin regulates lateral root initiation, root and stem elongation, and leaf expansion (for review, see Davies, 2004). Normal plant morphogenesis and environmental responses require modulation of auxin levels by controlling biosynthesis, regulating transport, and managing storage forms (for review, see Woodward and Bartel, 2005a). In some storage forms, the carboxyl group of IAA is conjugated to amino acids or peptides or to sugars, and free IAA can be released by hydrolases when needed (Bartel et al., 2001; Woodward and Bartel, 2005a). A second potential auxin storage form is the side chain-lengthened compound indole-3-butyric acid (IBA), which can be synthesized from IAA (Epstein and Ludwig-Müller, 1993) and is suggested to be shortened into IAA by peroxisomal β-oxidation (Bartel et al., 2001; Woodward and Bartel, 2005a).Genetic evidence suggests that the auxin activity of both IAA-amino acid conjugates and IBA requires free IAA to be released from these precursors (Bartel and Fink, 1995; Zolman et al., 2000). Mutation of Arabidopsis (Arabidopsis thaliana) genes encoding IAA-amino acid hydrolases, including ILR1, IAR3, and ILL2, reduces plant sensitivity to the applied IAA-amino acid conjugates that are substrates of these enzymes, including IAA-Leu, IAA-Phe, and IAA-Ala (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Rampey et al., 2004), which are present in Arabidopsis (Tam et al., 2000; Kowalczyk and Sandberg, 2001; Kai et al., 2007).Unlike the simple one-step release of free IAA from amino acid conjugates, release of IAA from IBA is suggested to require a multistep process (Zolman et al., 2007, 2008). Conversion of IBA to IAA has been demonstrated in a variety of plants (Fawcett et al., 1960; for review, see Epstein and Ludwig-Müller, 1993) and may involve β-oxidation of the four-carbon carboxyl side chain of IBA to the two-carbon side chain of IAA (Fawcett et al., 1960; Zolman et al., 2000, 2007). Mutation of genes encoding the apparent β-oxidation enzymes INDOLE-3-BUTYRIC ACID RESPONSE1 (IBR1), IBR3, or IBR10 results in IBA resistance, but does not alter IAA response or confer a dependence on exogenous carbon sources for growth following germination (Zolman et al., 2000, 2007, 2008), consistent with the possibility that these enzymes function in IBA β-oxidation but not fatty acid β-oxidation.Both conjugate hydrolysis and IBA β-oxidation appear to be compartmentalized. The IAA-amino acid hydrolases are predicted to be endoplasmic reticulum localized (Bartel and Fink, 1995; Davies et al., 1999) and enzymes required for IBA responses, including IBR1, IBR3, and IBR10, are peroxisomal (Zolman et al., 2007, 2008). Moreover, many peroxisome biogenesis mutants, such as peroxin5 (pex5) and pex7, are resistant to exogenous IBA, but remain IAA sensitive (Zolman et al., 2000; Woodward and Bartel, 2005b).Although the contributions of auxin transport to environmental and developmental auxin responses are well documented (for review, see Petrášek and Friml, 2009), the roles of various IAA precursors in these processes are less well understood. Expansion of root epidermal cells to control root architecture is an auxin-regulated process in which these roles can be dissected. Root epidermal cells provide soil contact and differentiate into files of either nonhair cells (atrichoblasts) or hair cells (trichoblasts). Root hairs emerge from trichoblasts as tube-shaped outgrowths that increase the root surface area, thus aiding in water and nutrient uptake (for review, see Grierson and Schiefelbein, 2002). Root hair length is determined by the duration of root hair tip growth, which is highly sensitive to auxin levels (for review, see Grierson and Schiefelbein, 2002). Mutants defective in the ABCG36/PDR8/PEN3 ABC transporter display lengthened root hairs and hyperaccumulate [³H]IBA, but not [³H]IAA, in root tip auxin transport assays (Strader and Bartel, 2009), suggesting that ABCG36 functions as an IBA effluxer and that IBA promotes root hair elongation. The related ABCG37/PDR9 transporter also can efflux IBA (Strader et al., 2008b; Růžička et al., 2010) and may have some functional overlap with ABCG36 (Růžička et al., 2010). In addition to lengthened root hairs, abcg36/pdr8/pen3 mutants display enlarged cotyledons, a second high-auxin phenotype. Both of these developmental phenotypes are suppressed by the mildly peroxisome-defective mutant pex5-1 (Strader and Bartel, 2009), suggesting that IBA contributes to cell expansion by serving as a precursor to IAA, which directly drives the increased cell expansion that underlies these phenotypes. However, whether IBA-derived IAA contributes to cell expansion events during development of wild-type plants is not known.Here, we directly demonstrate that peroxisome-defective mutants are defective in the conversion of IBA to IAA, consistent with previous reports that these genes are necessary for full response to applied IBA. We found that a mutant defective in three suggested IBA-to-IAA conversion enzymes displays low-auxin phenotypes, including decreased root hair expansion and decreased cotyledon size. We further found that these mutants suppress the long-root-hair and enlarged cotyledon phenotypes of an abcg36/pdr8 mutant, suggesting that endogenous IBA-derived IAA drives root hair and cotyledon expansion in wild-type seedlings.     

Enhancement of Enzymatic Synthesis of Citrate in vitro by Indole-3-Acetic Acid

Indoleacetic acid in physiological concentrations was shown to enhance the synthesis of citiate by purified citrate condensing enzyme from castor beans and pig heart. Michaelis constants reveal that with indoleacetic acid in the reaction mixture a higher concentration of acetyl-CoA was necessary to give maximal velocity. V values with indoleacetic acid in the reaction (physiological concentrations) exceeded V without indoleacetic acid in reaction. Citric acid synthesized from ¹⁴C acetyl CoA was highly radioactive when indoleacetie acid was present in the reaction, indicating that indoleacetic acid did in fact enhance the synthesis. The data were discussed from the point of view that these studies may provide the basis for studies directed at ultimate understanding of the mechanism of action of indoleacetic acid.     

Temperature-Sensitive Plant Cells with Shunted Indole-3-Acetic Acid Conjugation

Cells of henbane (Hyoscyamus muticus L.) grow indefinitely in culture without exogenous auxin. Cells of its temperature-sensitive variant XIIB2 grow like the wild type at 26[deg]C but die rapidly at 33[deg]C unless auxin is added to the medium. Despite this temperature-sensitive auxin auxotrophy, XIIB2 produces wild-type amounts of indole-3-acetic acid (IAA). IAA is the predominant auxin and is important for plant growth and development. Since the IAA production of the variant is functional, we investigated whether the synthesis or degradation of IAA metabolites, possibly active auxins themselves, is altered. The IAA metabolites were IAA-aspartate (IAAsp) and IAA-glucose. The wild type converted IAA mainly to IAAsp, whereas the variant produced mainly IAA-glucose. Exogenous auxin corrected the shunted IAA metabolism of the variant. The half-life of labeled IAAsp in the variant was reduced 21-fold, but in the presence of exogenous auxin it was not different from the wild type. The temperature sensitivity of XIIB2 was also corrected by supplying IAAsp. Pulse-chase experiments revealed that henbane rapidly metabolizes IAAsp to compounds not identical to IAA. The data show that the variant XIIB2 is a useful tool to study the function of IAA conjugates to challenge the popular hypothesis that IAA conjugates are merely slow-release storage forms of IAA.     

Biosynthesis of Indole-3-Acetic Acid by the Pine Ectomycorrhizal Fungus Pisolithus tinctorius

Previous work has indicated that anatomical and morphological changes (stunting and dichotomy) in roots of various conifers may be influenced by plant-growth-regulating substances secreted by mycorrhizae. Indole-3-acetic acid (IAA) has been tentatively identified as a major auxin produced by some selected ectomycorrhizae. We report the isolation and detection of IAA as a secondary metabolite from Pisolithus tinctorius by thin-layer chromatography, high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent (monoclonal antibody) assay (ELISA), and unequivocal identification by gas chromatography-mass spectrometry (GC-MS). The thin-layer chromatography methods for auxin isolation described here are novel, with the use of heptane-acetone-glacial acetic acid as the migrating solvent and formaldehyde, H(2)SO(4), and vanadate in detection. The acidic extract of the culture supernatant was methylated with ethereal diazomethane to detect IAA as methyl-3-IAA by HPLC, ELISA, and GC-MS. The quantitative amount of IAA detected ranged from 4 to 5 mumol liter by HPLC and ELISA. Another unidentified metabolite was detected by GC-MS with a typical indole nucleus (m/z = 130), indicating that it could be an intermediate in auxin metabolism. Plant response (Pseudotsuga menziesii, Douglas fir) was monitored upon inoculation of P. tinctorius and l-tryptophan. There was a consistent increase in plant height and stem diameter as a result of the two treatments, with statistical differences in dry weights of the shoots and roots.     

Indole-3-acetic Acid Levels of Plant Tissue as Determined by a New High Performance Liquid Chromatographic Method

A method for the analysis of indole-3-acetic acid (IAA) in plant extracts has been developed based on high performance liquid chromatography separation of IAA on a microparticulate strong anion exchange column followed by quantitation with two selective detectors: an electrochemical, carbon paste amperometric detector and/or a fluorescence detector. The detection limit for IAA is less than 1 nanogram with the fluorescence detector and less than 50 picograms with the electrochemical detector.

The IAA levels are reported for various tissues of wheat, pinto beans, soybeans, cotton, and corn.