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.     

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.     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Identification and characterization of an indigo-producing oxygenase involved in indole 3-acetic acid utilization by Acinetobacter baumannii

Acinetobacter baumannii harbours a gene cluster similar to the iac locus of Pseudomonas putida 1290, which can catabolize the plant hormone indole 3-acetic acid (IAA) as an energy source. However, there has been no evidence showing that IAA can be utilized by A. baumannii. This study showed that A. baumannii can grow in M9 minimal medium containing IAA as the sole carbon source. A mutagenesis study indicated that iacA, encoded in the iac locus of A. baumannii, is involved in the catabolism of IAA. As shown by western blotting analysis, the IacA protein was detected in A. baumannii grown in M9 minimal medium with IAA but not with pyruvate, suggesting that the expression of iacA is regulated by the presence of IAA. In vitro studies have shown that IacA can oxidize indole, an IAA-like molecule, converting it to indoxyl, which spontaneously dimerises to form indigo. In this study, we show that the crude extracts from either wild-type A. baumannii or Escherichia coli overexpressing IacA can oxidize IAA. These results imply that the iac gene cluster of A. baumannii is involved in IAA degradation and that the iacA gene is upregulated when cells encounter IAA in their native environments.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.Abbreviations 4Cl-IAA 4-chloro-indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA Indole-3-acetic acid - IAA-C5-BSA, IAA-C1-BSA, IAA-NI-BSA hapten-carrier-conjugates with IAA linked to bovine serum albumin through the C5-position, the carboxyl group, and the indole nitrogen, respectively - IAA-Me the methylester of IAA This study was supported by the Danish Research Council (SJVF 13-4148 and 13-4547 to P.U.) and by The Research Center for Plant Biotechnology.     

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.     

Activity of 1,2-benzisoxazole-3-one and indole-2,3-dione on plant regeneration in vitro and on cell elongation

We tested the morphogenetic and cell elongating activity of 1,2-benzisoxazole-3-one, a compound similar to 1,2-benzisoxazole-3-acetic acid but lacking the lateral carbon chain. For comparison, we tested also the activity of indole 2,3-dione, having the same indolic ring as indole 3-acetic acid but no lateral carbon chain. The tests were made on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. We found that 1,2 benzisoxazole-3-one retains part of the high shoot inducing activity of 1,2-benzisoxazole-3-aceticacid, while indole-2,3-dione is inactive. Both compounds have no effect on root induction or cell elongation. It seems therefore that the activity of 1,2 benzisoxazole-3-acetic acid is partly related to the structure of its ring, and that also in this respect 1,2 benzisoxazole-3-acetic acid differs from other auxinlike compounds.Abbreviations BOA 1,2-benzisoxazole-3-acetic acid - BOO 1,2-benzisoxazole-3-one - IAA in-dole-3-acetic acid     

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1-28 microg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 microg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 microg IAA/ml respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 microg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a "root brush" with location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.     

The indole alkaloids brucine, yohimbine, and hypaphorine are indole-3-acetic acid-specific competitors which do not alter auxin transport

The indole alkaloids brucine and yohimbine, just like hypaphorine, counteract indole-3-acetic acid (IAA) activity in seedling roots, root hairs and shoots, but do not appear to alter auxin transport in roots or in cultured cells. In roots, the interactions between IAA and these three alkaloids appear competitive and specific since these molecules interact with IAA but with neither 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), two synthetic auxins. The data reported further support the hypothesis that hypaphorine brucine and yohimbine compete with IAA on some auxin-binding proteins likely to be auxin receptors and that 2,4-D and NAA are not always perceived by the same receptor as IAA or the same component of that receptor. At certain steps of plant development and in certain cells, endogenous indole alkaloids could be involved in IAA activity regulation together with other well-described mechanisms such as conjugation or degradation. Hypaphorine with other active indole alkaloids remaining to be identified, might be regarded as a new class of IAA antagonists.     

Identification of indole compounds secreted byFrankia HFPArI3 in defined culture medium

Indole compounds secreted byFrankia sp. HFPArI3 in defined culture medium were identified with gas chromatography-mass spectrometry (GC-MS). WhenFrankia was grown in the presence of¹³C(ring-labelled)-L-tryptophan,¹³C-labelled indole-3-acetic acid (IAA), indole-3-ethanol (IEtOH), indole-3-lactic acid (ILA), and indole-3-methanol (IMeOH) were identified.High performance liquid chromatography (HPLC) and GC-MS with selected ion monitoring were used to quantify levels of IAA and IEtOH inFrankia culture medium. IEtOH was present in greater abundance than IAA in every experiment. When no exogenous trp was supplied, no or only low levels of indole compounds were detected.Seedling roots ofAlnus rubra incubated in axenic conditions in the presence of indole-3-ethanol formed more lateral roots than untreated plants, indicating that IEtOH is utilized by the host plant, with physiological effects that modify patterns of root primordium initiation.     

Photoproduction of indole 3-acetic acid by Rhodobacter sphaeroides from indole and glycine

Rhodobacter sphaeroides photoproduced indole 3-acetic acid (IAA) when the precursor L-tryptophan was added to the basal medium. Of the other organic carbon sources that influenced IAA formation from L-tryptophan, -ketoglutaric acid gave the maximum formation of 530 mg 1^–1 in less than 24 h of illuminated anaerobic incubation. IAA was also produced from indole, glycine and glucose together by Rb. sphaeroides under photoanaerobic conditions yielding 61 mg l^–1 within 30 fh.     

Auxin production by plant-pathogenic pseudomonads and xanthomonads

Pathogenic strains of Xanthomonas campestris pv. glycines which cause hypertrophy of leaf cells of susceptible soybean cultivars and nonpathogenic strains which do not cause hypertrophy were compared for their ability to produce indole compounds, including the plant hormone indole-3-acetic acid (IAA) in liquid media with or without supplementation with l-tryptophan. Several additional strains of plant-pathogenic xanthomonads and pseudomonads were also tested for IAA production to determine whether in vitro production of IAA is related to the ability to induce hypertrophic growth of host tissues. Indoles present in culture filtrates were identified by thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy, mass spectroscopy, and gas chromatography-mass spectrometry and were quantitated by high-performance liquid chromatography. All strains examined produced IAA when liquid media were supplemented with l-tryptophan. The highest levels of IAA were found in culture filtrates from the common bean pathogen Pseudomonas syringae pv. syringae, and this was the only bacterium tested which produced IAA without addition of tryptophan to the medium. Additional indoles identified in culture filtrates of the various strains included indole-3-lactic acid, indole-3-aldehyde, indole-3-acetamide, and N-acetyltryptophan. Pseudomonads and xanthomonads could be distinguished by the presence of N-acetyltryptophan, which was found only in xanthomonad culture filtrates.     

Indole compounds released by the ectendomycorrhizal fungal strain MrgX isolated from a pine nursery

The production and metabolism of indole compounds in pure cultures of the ectendomycorrhizal strain MrgX, a common symbiont of Scots pine in forest nurseries, were investigated. Different indole compounds produced by this fungus were purified and identified by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. Indole-3-acetic acid (IAA) and indole-3-carboxylic acid were the most abundant. Although MrgX is able to synthesize IAA when cultivated on a medium without tryptophan, much higher IAA production was obtained when 1 mM tryptophan was added. Buffering of the medium at pH 5.8 was shown to be essential for IAA accumulation in the culture filtrate. In vitro IAA-synthesizing activity of the enzymes extracted from the mycelia of MrgX was also maximal when mycelia were grown in a buffered, tryptophan-supplemented medium. The hydrogen ion concentration strongly affected in vivo activity of IAA-synthesizing enzymes. This activity was rather weak at acid pH and was stimulated by increase in pH up to 8.5. These results and their possible significance for ectendo-mycorrhizal symbiosis are discussed with reference to the hormonal metabolism of ectomycorrhizal fungi and ectomycorrhizae.     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Oxygen-dependent catabolism of indole-3-acetic acid in Bradyrhizobium japonicum.

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid (IAA). Examination of this catabolism in strain 110 by in vivo experiments has revealed an enzymatic activity catalyzing the degradation of IAA and 5-hydroxy-indole-3-acetic acid. The activity requires addition of the substrates for induction and is oxygen dependent. The highest activity is obtained when the concentration of inducer is 0.2 mM. Spectrophotometric data are consistent with the suggestion that the indole ring is broken during degradation of IAA. We hypothesize that the enzyme catalyzes an oxygen-consuming opening of the indole ring analogous to the one catalyzed by tryptophan 2,3-dioxygenase. The pattern of metabolite usage by known tryptophan-auxotrophic mutants and studies of metabolites by high-performance liquid chromatography indicate that anthranilic acid is a terminal degradation product in the proposed pathway.     

Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression

Rhizospheric bacterial strains are known to produce indole-3-acetic acid (IAA) through different pathways, and such IAA may be beneficial to plants at low concentrations. IAA biosynthesis by a natural isolate of Azospirillum brasilense SM was studied and observed to be tryptophan-inducible and -dependent in nature. While our work demonstrated the operation of the indole pyruvic acid pathway, the biochemical and molecular evidence for the genes of the indole acetamide (IAM) pathway were lacking in A. brasilense SM. This led us to use the IAM pathway genes as targets for metabolic engineering, with the aim of providing an additional pathway of IAA biosynthesis and improving IAA levels in A. brasilense SM. The introduction of the heterologous IAM pathway, consisting of the iaaM and iaaH genes, not only increased the IAA levels by threefold but also allowed constitutive expression of the same genes along with efficient utilization of IAM as a substrate. Such an engineered strain showed a superior effect on the lateral branching of sorghum roots as well as the dry weight of the plants when compared with the wild-type strain. Such an improved bioinoculant could be demonstrated to enhance root proliferation and biomass productivity of treated plants compared with the parental strain.