Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.
ABSTRACT: Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.
Project description:The plant growth-promoting rhizobacterium Enterobacter cloacae UW5 synthesizes the plant growth hormone indole-3-acetic acid (IAA) via the indole-3-pyruvate pathway utilizing the enzyme indole-3-pyruvate decarboxylase that is encoded by ipdC. In this bacterium, ipdC expression and IAA production occur in stationary phase and are induced by an exogenous source of tryptophan, conditions that are present in the rhizosphere. The aim of this study was to identify the regulatory protein that controls the expression of ipdC. We identified a sequence in the promoter region of ipdC that is highly similar to the recognition sequence for the Escherichia coli regulatory protein TyrR that regulates genes involved in aromatic amino acid transport and metabolism. Using a tyrR insertional mutant, we demonstrate that TyrR is required for IAA production and for induction of ipdC transcription. TyrR directly induces ipdC expression, as was determined by real-time quantitative reverse transcription-PCR, by ipdC promoter-driven reporter gene activity, and by electrophoretic mobility shift assays. Expression increases in response to tryptophan, phenylalanine, and tyrosine. This suggests that, in addition to its function in plant growth promotion, indolepyruvate decarboxylase may be important for aromatic amino acid uptake and/or metabolism.
Project description:Plants, bacteria and some fungi are known to produce indole-3-acetic acid (IAA) by employing various pathways. Among these pathways, the indole-3-pyruvic acid (IPA) pathway is the best studied in green plants and plant-associated beneficial microbes. While IAA production circuitry in plants has been studied for decades, little is known regarding the IAA biosynthesis pathway in fungal species. Here, we present the first data for IAA-producing genes and the associated biosynthesis pathway in a non-pathogenic fungus, Neurospora crassa. For this purpose, we used a computational approach to determine the genes and outlined the IAA production circuitry in N. crassa. We then validated these data with experimental evidence. Here, we describe the homologous genes that are present in the IPA pathway of IAA production in N. crassa. High-performance liquid chromatography and thin-layer chromatography unambiguously identified IAA, indole-3-lactic acid (ILA) and tryptophol (TOL) from cultures supplemented with tryptophan. Deletion of the gene (cfp) that encodes the enzyme indole-3-pyruvate decarboxylase, which converts IPA to indole-3-acetaldehyde (IAAld), results in an accumulation of higher levels of ILA in the N. crassa culture medium. A double knock-out strain (?cbs-3;?ahd-2) for the enzyme IAAld dehydrogenase, which converts IAAld to IAA, shows a many fold decrease in IAA production compared with the wild type strain. The ?cbs-3;?ahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.
Project description:IAA biosynthetic pathways in a basidiomycetous yeast, Rhodosporidiobolus fluvialis DMKU-CP293, were investigated. The yeast strain showed tryptophan (Trp)-dependent IAA biosynthesis when grown in tryptophan supplemented mineral salt medium. Gas chromatography-mass spectrometry was used to further identify the pathway intermediates of Trp-dependent IAA biosynthesis. The results indicated that the main intermediates produced by R. fluvialis DMKU-CP293 were tryptamine (TAM), indole-3-acetic acid (IAA), and tryptophol (TOL), whereas indole-3-pyruvic acid (IPA) was not found. However, supplementation of IPA to the culture medium resulted in IAA peak detection by high-performance liquid chromatography analysis of the culture supernatant. Key enzymes of three IAA biosynthetic routes, i.e., IPA, IAM and TAM were investigated to clarify the IAA biosynthetic pathways of R. fluvialis DMKU-CP293. Results indicated that the activities of tryptophan aminotransferase, tryptophan 2-monooxygenase, and tryptophan decarboxylase were observed in cell crude extract. Overall results suggested that IAA biosynthetic in this yeast strain mainly occurred via the IPA route. Nevertheless, IAM and TAM pathway might be involved in R. fluvialis DMKU-CP293.
Project description:Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis is characterized by excessive host tumour formation. U. maydis is able to produce indole-3-acetic acid (IAA) efficiently from tryptophan. To assess a possible connection to the induction of host tumours, we investigated the pathways leading to fungal IAA biosynthesis. Besides the previously identified iad1 gene, we identified a second indole-3-acetaldehyde dehydrogenase gene, iad2. Deltaiad1Deltaiad2 mutants were blocked in the conversion of both indole-3-acetaldehyde and tryptamine to IAA, although the reduction in IAA formation from tryptophan was not significantly different from Deltaiad1 mutants. To assess an influence of indole-3-pyruvic acid on IAA formation, we deleted the aromatic amino acid aminotransferase genes tam1 and tam2 in Deltaiad1Deltaiad2 mutants. This revealed a further reduction in IAA levels by five- and tenfold in mutant strains harbouring theDeltatam1 andDeltatam1Deltatam2 deletions, respectively. This illustrates that indole-3-pyruvic acid serves as an efficient precursor for IAA formation in U. maydis. Interestingly, the rise in host IAA levels upon U. maydis infection was significantly reduced in tissue infected with Deltaiad1Deltaiad2Deltatam1 orDeltaiad1Deltaiad2Deltatam1Deltatam2 mutants, whereas induction of tumours was not compromised. Together, these results indicate that fungal IAA production critically contributes to IAA levels in infected tissue, but this is apparently not important for triggering host tumour formation.
Project description:The plant growth-promoting rhizobacteria (PGPR) strain Bacillus amyloliquefaciens SQR9, isolated from the cucumber rhizosphere, protects the host plant from pathogen invasion and promotes plant growth through efficient root colonization. The phytohormone indole-3-acetic acid (IAA) has been suggested to contribute to the plant-growth-promoting effect of Bacillus strains. The possible IAA synthetic pathways in B. amyloliquefaciens SQR9 were investigated in this study, using a combination of chemical and genetic analysis.Gene candidates involved in tryptophan-dependent IAA synthesis were identified through tryptophan response transcriptional analysis, and inactivation of genes ysnE, dhaS, yclC, and yhcX in SQR9 led to 86, 77, 55, and 24 % reductions of the IAA production, respectively. The genes patB (encoding a conserved hypothetical protein predicted to be an aminotransferase), yclC (encoding a UbiD family decarboxylase), and dhaS (encoding indole 3-acetaldehyde dehydrogenase), which were proposed to constitute the indole-3-pyruvic acid (IPyA) pathway for IAA biosynthesis, were separately expressed in SQR9 or co-expressed as an entire IAA synthesis pathway cluster in SQR9 and B. subtilis 168, all these recombinants showed increased IAA production. These results suggested that gene products of dhaS, patB, yclB, yclC, yhcX and ysnE were involved in IAA biosynthesis. Genes patB, yclC and dhaS constitute a potential complete IPyA pathway of IAA biosynthesis in SQR9.In conclusion, biosynthesis of IAA in B. amyloliquefaciens SQR9 occurs through multiple pathways.
Project description:Indole-3-acetic acid (IAA) is an imperative phytohormone for plant growth and development. Ectomycorrhizal fungi (ECM) are able to produce IAA. However, only a few studies on IAA biosynthesis pathways in ECM fungi have been reported. This study aimed to investigate the IAA biosynthesis pathway of six ECM cultures including Astraeus odoratus, Gyrodon suthepensis, Phlebopus portentosus, Pisolithus albus, Pisolithus orientalis and Scleroderma suthepense. The results showed that all ECM fungi produced IAA in liquid medium that had been supplemented with L-tryptophan. Notably, fungal IAA levels vary for different fungal species. The detection of indole-3-lactic acid and indole-3-ethanol in the crude culture extracts of all ECM fungi indicated an enzymatic reduction of indole-3-pyruvic acid and indole-3-acetaldehyde, respectively in the IAA biosynthesis via the indole-3-pyruvic acid pathway. Moreover, the tryptophan aminotransferase activity confirmed that all ECM fungi synthesize IAA through the indole-3-pyruvic acid pathway. Additionally, the elongation of rice and oat coleoptiles was stimulated by crude culture extract. This is the first report of the biosynthesis pathway of IAA in the tested ECM fungi.
Project description:Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.
Project description:Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg(-1)ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity.
Project description:The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.
Project description:Arthrobacter pascens ZZ21 is a plant-beneficial, fluoranthene-degrading bacterial strain found in the rhizosphere. The production of the phytohormone indole-3-aectic acid (IAA) by ZZ21 is thought to contribute to its ability to promote plant growth and remediate fluoranthene-contaminated soil. Using genome-wide analysis combined with metabolomic and high-performance liquid chromatography-mass spectrometry (HPLC-MS) analyses, we characterized the potential IAA biosynthesis pathways in A. pascens ZZ21. IAA production increased 4.5-fold in the presence of 200 mg·L-1 tryptophan in the culture medium. The transcript levels of prr and aldH, genes which were predicted to encode aldehyde dehydrogenases, were significantly upregulated in response to exogenous tryptophan. Additionally, metabolomic analysis identified the intermediates indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and the enzymatic reduction product of the latter, indole-3-lactic acid (ILA), among the metabolites of ZZ21, and subsequently also IAM, ILA, and indole-3-ethanol (TOL), which is the enzymatic reduction product of indole-3-acetaldehyde, by HPLC-MS. These results suggest that the tryptophan-dependent IAM and IPyA pathways function in ZZ21.