Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by Pseudomonas putida Strain 1290

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.     

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.     

Production of the Phytohormone Indole-3-Acetic Acid by Estuarine Species of the Genus Vibrio

Strains of Vibrio spp. isolated from roots of the estuarine grasses Spartina alterniflora and Juncus roemerianus produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA, recA, and rpoA genes was used for phylogenetic analysis of environmental isolates within the genus Vibrio. Eight Vibrio type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing Vibrio species. Six additional environmental isolates similar to four of the Vibrio type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment.Estuaries along the east coast of temperate North America are ecologically valuable, productive systems dominated by only a few species of plants. Spartina alterniflora (smooth cord grass; hereinafter referred to as Spartina) is a keystone species responsible for very high rates of primary production in Atlantic coast marshes and is a major contributor to the global cycling of several elements (10, 14, 15, 35, 38, 39, 45). Juncus roemerianus (black needle rush; hereinafter referred to as Juncus) is a common subdominant species (28) residing in areas of higher elevation, lower salinity, and less frequent tidal inundation. The roots of these macrophytes are associated with a diverse assemblage of microorganisms, including N₂-fixing and sulfate-reducing bacteria, which greatly contribute to their productivity (30, 31).The phytohormone indole-3-acetic acid (IAA) is the most commonly occurring naturally produced auxin and the most thoroughly studied plant growth regulator. IAA directs several aspects of plant growth and development (37), including the induction and regulation of a variety of processes: e.g., cell division, root extension, vascularization, apical dominance, and tropisms (6, 32). The effects of IAA on plant root tissue are concentration dependent and can be species specific. Responses to increasing IAA concentrations advance from the stimulation of primary root tissue to the development of lateral and adventitious roots and finally to the complete cessation of root growth (1, 6, 16, 29, 32, 37, 44).Many microorganisms interact with and affect their environment through the production and transudation of signal compounds (17). The findings of numerous studies (see, e.g., references 8, 23, 25, and 37) demonstrate that a variety of plant-associated terrestrial bacteria produce and exude IAA. Auxin synthesis by cyanobacteria has also been reported previously (40). IAA is thought to reduce the integrity of plant cell walls by upregulating the production of cellulases and hemicelluloses, resulting in the leakage of some simple sugars and other nutrients that would benefit root-associated microorganisms (17). Likewise, root growth would be an advantage to resident bacteria due to the increased availability of root exudates and root surface for growth. Microorganisms that produce IAA can influence the host plant and function as pathogens, symbionts, or growth regulators, depending on how their IAA production influences the concentration of the plant''s endogenous IAA pool and on the sensitivity of the plant to auxin. Organisms such as Erwinia chrysanthemi, Pseudomonas savastanoi, and Agrobacterium tumefaciens are phytopathogens of many host plants (11, 21, 23, 46). Other organisms, including Azospirillum brasilense and Pseudomonas putida GR12-2, have proven beneficial to plants, and many IAA producers have been shown to stimulate increases in root mass and/or length (20, 37, 44).The aim of the present study was to assess IAA synthesis by Vibrio strains isolated from the roots of highly productive salt marsh grasses. The Salkowski assay was used to perform an initial screening for the presence of IAA, gas chromatography-mass spectroscopy (GC-MS) verified and quantified IAA production, and multilocus sequence typing (MLST) analysis classified all isolates within the genus Vibrio.     

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.     

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

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.     

Contribution of Indole-3-Acetic Acid Production to the Epiphytic Fitness of Erwinia herbicola

Erwinia herbicola 299R produces large quantities of indole-3-acetic acid (IAA) in culture media supplemented with l-tryptophan. To assess the contribution of IAA production to epiphytic fitness, the population dynamics of the wild-type strain and an IAA-deficient mutant of this strain on leaves were studied. Strain 299XYLE, an isogenic IAA-deficient mutant of strain 299R, was constructed by insertional interruption of the indolepyruvate decarboxylase gene of strain 299R with the xylE gene, which encodes a 2,3-catechol dioxygenase from Pseudomonas putida mt-2. The xylE gene provided a useful marker for monitoring populations of the IAA-deficient mutant strain in mixed populations with the parental strain in ecological studies. A root bioassay for IAA, in which strain 299XYLE inhibited significantly less root elongation than strain 299R, provided evidence that E. herbicola produces IAA on plant surfaces in amounts sufficient to affect the physiology of its host and that IAA production in strain 299R is not solely an in vitro phenomenon. The epiphytic fitness of strains 299R and 299XYLE was evaluated in greenhouse and field studies by analysis of changes in the ratio of the population sizes of these two strains after inoculation as mixtures onto plants. Populations of the parental strain increased to approximately twice those of the IAA-deficient mutant strain after coinoculation in a proportion of 1:1 onto bean plants in the greenhouse and onto pear flowers in field studies. In all experiments, the ratio of the population sizes of strain 299R and 299XYLE increased during periods of active growth on plant tissue but not when population sizes were not increasing with time.

Many plant-associated bacteria have the ability to produce the plant growth regulator indole-3-acetic acid (IAA) (5, 9, 25, 33). IAA is involved in diseases caused by gall- and knot-forming bacterial species (33); however, its role in other bacteria remains undefined. It is unclear whether these bacteria produce IAA during colonization of plant surfaces and whether this metabolite is beneficial to the bacteria during their growth and survival in the phyllosphere. The production of IAA may enable bacteria to detoxify tryptophan analogues present on plant surfaces (15), to downregulate genes involved in plant defense responses (33), or to inhibit the development of the hypersensitive response by plants (26). We recently demonstrated that the ipdC gene, which encodes the indolepyruvate decarboxylase of Erwinia herbicola (Pantoea agglomerans) 299R and which is involved in the indolepyruvate pathway for IAA synthesis in this epiphytic strain (2), is osmoresponsive and plant inducible (3). We hypothesized that the secretion of IAA may modify the microhabitat of epiphytic bacteria by increasing nutrient leakage from plant cells; enhanced nutrient availability may better enable IAA-producing bacteria to colonize the phyllosphere and may contribute to their epiphytic fitness (1).Few studies have attempted to determine the ecological significance of IAA production in pathogenic bacteria. Varvaro and Martella (31) showed that IAA-deficient mutants of Pseudomonas syringae pv. savastanoi, obtained by selection for resistance to α-methyltryptophan, were reduced in their ability to colonize and survive on olive leaf surfaces. The survival of an α-methyltryptophan-resistant IAA-deficient mutant of P. syringae pv. savastanoi in knots also was affected, its population declining more rapidly than that of the parental strain when inoculated alone into oleander leaf tissue (28). The importance of IAA production in bacterial colonization of bean leaves was also tested with the brown spot pathogen P. syringae pv. syringae and an IAA-deficient mutant derived by insertional mutagenesis (21). Although no difference in the survival of the parental and mutant strains on bean leaves was observed in the greenhouse, a small difference in their behavior was apparent in experiments conducted in a mist chamber (21). There have been no studies of the role of IAA production in plant-associated bacteria that do not cause disease.IAA biosynthesis is not essential for bacterial growth and survival, since IAA-deficient mutants grow as well as their IAA-producing parental strain in vitro (2, 29). Large differences in the epiphytic behaviors of IAA-producing bacteria and isogenic IAA-deficient mutants consequently would not be expected. Even small contributions of IAA production to epiphytic fitness could account for the common presence of this phenotype in epiphytic bacteria (19). Measurements of changes in the ratio of two strains following coinoculation, a common approach in ecological studies, can allow the detection of even small differences in the competitive behaviors of two organisms. This approach can detect much smaller differences in behavior between closely related species than comparison of populations of these species when present singly in separate habitats (16). In this study, we tested the role of IAA in the epiphytic fitness of E. herbicola by comparing the relative changes in the population sizes of the parental and IAA-deficient mutant strains with time after their inoculation onto plants in both controlled and field environments.     

Identification of Indole-3-Acetic Acid in the Basidiomycete Schizophyllum commune

Indole-3-acetic acid (IAA) was detected in the ether extracts of culture filtrates of indigotin-producing strains of the basidiomycete Schizophyllum commune. Several solvents, known to give distinctly different R_F values for IAA, and 3 location reagents gave identical results with synthetic IAA and IAA found in the extract. Confirmation was obtained by the Avena straight growth test, split pea test, and ultraviolet absorption spectrum.     

Production of Indole-3-Acetic Acid by Bradyrhizobium japonicum: A Correlation with Genotype Grouping and Rhizobitoxine Production

Bioassays show that rhizobitoxine-producing strains of Bradyrhizobiumjaponicum excreted another phytotoxic compound into their culturefluid. This compound was purified and identified by HPLC andmass spectrometry as indole-3-acetic acid (IAA). The levelsof IAA produced by the different strains of B. japonicum, forwhich the genotype groups have been determined with respectto the degree of base substitution in and around nifDKE, werequantified using gas chromatography/mass spectrometry and adeuterated internal standard. Genotype II strains, which producerhizobitoxine, excreted more than 20µof IAA into theirculture fluid. However, no IAA was detected in the culture supernatantsof genotype I strains, which do not produce rhizobitoxine. Thiswas true even when tryptophan was added to the medium. Moreover,cells of genotypes I and II strains, which were grown underour culture conditions, did not show IAA degradation activity.These results suggest that, in wild-type B. japonicum strains,complete IAA biosynthesis is confined exclusively to genotypeII strains that produce rhizobitoxine. (Received April 9, 1990; Accepted October 6, 1990)     

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.     

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)     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

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.     

The Identification of Indole-3-Acetic Acid and Indole-3-Acetamide in the Hypocotyls of Japanese Cherry

Both indole-3-acetamide (IAM) and indole-3-acetic acid (IAA)were identified in extracts of the hypocotyls of Japanese cherryby GC/MS. Exogenous IAA and IAM promoted the elongation of segmentsof these hypocotyls and the effect of IAA applied together withIAM was the same as that of IAA alone. (Received July 29, 1992; Accepted October 19, 1992)     

Indole-3-Acetic Acid Production by Endophytic Streptomyces sp. En-1 Isolated from Medicinal Plants

Plant-associated actinobacteria are rich sources of bioactive compounds including indole-derived molecules such as phytohormone indole-3-acetic acid (IAA). In view of few investigations concerning the biosynthesis of IAA by endophytic actinobacteria, this study evaluated the potential of IAA production in endophytic streptomycete isolates sourced from medicinal plant species Taxus chinensis and Artemisia annua. By HPLC analysis of IAA combined with molecular screening approach of iaaM, a genetic determinant of streptomycete IAA synthesis via indole-3-acetamide (IAM), our data showed the putative operation of IAM-mediated IAA biosynthesis in Streptomyces sp. En-1 endophytic to Taxus chinensis. Furthermore, using the co-cultivation system of model plant Arabidopsis thaliana and streptomycete, En-1 was found to be colonized intercellularly in the tissues of Arabidopsis, an alternative host, and the effects of endophytic En-1 inoculation on the model plant were also assayed. The phytostimulatory effects of En-1 inoculation suggest that IAA-producing Streptomyces sp. En-1 of endophytic origin could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.     

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.     

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.     

Occurrence of Indole-3-Acetic Acid in Buds of Pinus silvestris

The major ether-soluble, growth-stimulating substance detected by the Avena coleoptile straight-growth test in extract from sprouting buds of Scots pine (Pinus silvestris L.) was identified as indole-3-acetic acid by Rf values in 5 solvent systems and by its elution volume in ethanol on a Sephadex LH-20 column. When the substance was applied to the growth solution of wheat roots in a special test the growth in length of the roots was at first inhibited, but growth was recovered after about 6 hours in the same manner as when small quantities of IAA were applied. The extracts also contained large amounts of growth inhibitors which interfered with the auxin response if they were not removed.     

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.