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.     

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.     

Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase.

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for less than or equal to 1.5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6.8) supplemented with 10 mM-L-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6.0) containing 1 mM-L-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH approximately 7 or pH approximately 6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.     

Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla.

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.     

Some tryptophan pathways in the phytopathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv.oryzae, the causal or-ganism of bacterial blight of rice which produces leaf blight as well as kresek (wilt) symptoms in plants were tested for indole, auxin production in culture supplemented withl-tryptophan. On the basis of indoleacetic acid (IAA) production the isolates were grouped into IAA-positive and IAA-negative. Out of 17 isolates, 11 were IAA-positive while 6 were IAA-negative. The isolates metabolized tryptophan through two different routes and the isolates vary in the pathway of tryptophan utilization. The IAA-positive isolates converted tryptophan to IAA as the end product, whereas the IAA-negative isolates formed anthranilate as an intermediate metabolite and finally produced pyrocatecholvia the kynurenine pathway. Quantification of tryptophan metabolism revealed that the maximum production of IAA and pyrocatechol in culture occurred during 2-d incubation at 30±2°C.     

Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti.

Bacterial indoleacetic acid (IAA) production, which has been proposed to play a role in the Rhizobium-legume symbiosis, is a poorly understood process. Previous data have suggested that IAA biosynthesis in Rhizobium meliloti can occur through an indolepyruvate intermediate derived from tryptophan by an aminotransferase activity. To further examine this biosynthetic pathway, the aromatic aminotransferase (AAT) activity of Rhizobium meliloti 102F34 (F34) was characterized. At least four proteins were detected on nondenaturing gels of F34 protein extracts that exhibited AAT activity. All four of these AATs were constitutively produced and utilized the aromatic amino acids tryptophan, phenylalanine, and tyrosine as amino substrates. Two AATs were also capable of using aspartate. Plasmids from an F34 gene bank were identified that coded for the synthesis of at least three of these proteins, and the respective gene sequences were localized by transposon mutagenesis. Selected transposon insertions were recombined into the F34 genome to produce strains defective in two of these proteins (AAT1 and AAT2). Characterization of the mutants revealed that neither was essential for the biosynthesis of IAA in the absence of exogenous tryptophan, but that both contributed to IAA biosynthesis when high levels of exogenous tryptophan were present. AAT1 and AAT2 were also not required for the production of a minimal level of aromatic amino acids, but both were able to scavenge nitrogen from the aromatic amino acids during nitrogen deprivation. Neither AAT1 nor AAT2 was essential for symbiosis with alfalfa.     

Indole-3-acetic acid biosynthesis in Lemna gibba studied using stable isotope labeled anthranilate and tryptophan

IAA biosynthesis in many plants, including Lemna gibba, has been shown to involve at least two different pathways, one from tryptophan and a tryptophan-independent route. To study the kinetics of IAA biosynthesis in Lemna, we simultaneously measured the incorporation of label from [15N]-anthranilate and [2H5]-tryptophan into IAA by Lemna plants in short term feeding studies. The data show that label from anthranilate rapidly goes into IAA and tryptophan. Labeling of the IAA pool by [15N]-anthranilate slightly precedes labeling of the tryptophan pool, confirming that more than one route to IAA exists in these plants. Longer term feeding studies (5–25 h) suggest that exogenous tryptophan is used preferentially to label IAA as compared to tryptophan made by the plant. This is indicated by the fact that the IAA pool was more enriched than the tryptophan pool in [2H5]-label, but less enriched than the tryptophan pool in [15N] (which comes about by de novo synthesis of tryptophan from [15N]-anthranilate by the plant).     

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.     

Production and metabolism of indole acetic acid in roots and root nodules of Phaseolus mungo

The mature root nodules of Phaseolus mungo (L.), a leguminous pulse, contain higher amount of indole acetic acid (IAA) than non-nodulated roots. The tryptophan pool present in the mature nodule and young roots might serve as a precursor for the IAA production. Presence of IAA metabolising enzymes – IAA oxidase and peroxidase – indicate the metabolism of IAA in the nodules and roots. In culture, the symbiont, isolated from the nodules, produced a high amount of IAA, when tryptophan was supplied in the medium as a precursor. The symbiont preferred l-isomer over the dl- or d-isomer of tryptophan for IAA production.The important physiological implication of the IAA production in the legume-Rhizobium symbiosis is discussed.     

Studies on plant growth substances,IAA metabolism and nitrogenase activity in root nodules of phaseolus aureus Roxb. var.mungo

The mature nodules ofPhaseolus aureus Roxb. var.mungo possessed, in comparison with young and old nodules, higher activities of nitrogenase (N_2ase), and indol-3-ylacetic acid (IAA) metabolic enzymes like IAA oxidase, methylene oxindole reductase and peroxidase; higher levels of IAA-like, gibberellic acid-like (GA), and cytokinin-like (CK) substances and tryptophan, and lower level of phenol. The abscisic acid-like (ABA) substance level was higher in the old nodules. The N_2ase activity in the mature nodules changed parallelly with IAA and OK. butoppositely with GA and ABA. The changes in tryptophan level, IAA oxidizing enzymes, and phenol metabolism controlled the IAA level in the nodules. The nodules developed similarly throughout the year, but they had variable hormone levels in different seasons. This indicated that the formation and growth of the nodules was controlled not only by the nodular hormones.     

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.     

Construction of IAA-Deficient Mutants of Pseudomonas moraviensis and Their Comparative Effects with Wild Type Strains as Bio-inoculant on Wheat in Saline Sodic Soil

Tryptophan role in microbial biosynthesis of Indole Acetic Acid (IAA) is very distinct. In present study IAA producing bacteria Pseudomonas moraviensis was applied on wheat for improving growth and physiology; in the presence or absence of L-tryptophan in saline sodic field. Aqueous solution of tryptophan was added to the rhizosphere soil at 10?mg L^?1 with irrigated water. The survival efficiency of P. moraviensis measured in the presence of NaCl and mixture of salts. P. moraviensis increased P, NO₃–N and K contents in soil by 18–35% and further 12–15% increase was recorded in the presence of tryptophan. There were 40–80% increases in indole acetic acid (IAA), abscisic acid (ABA) and gibberellic acid (GA) contents of rhizosphere soil, and 40–45% increase in leaves when tryptophan was added with P. moraviensis. In the second phase, IAA deficient mutants of P. moraviensis were constructed and tested for the conversion of tryptophan to IAA. In transposon mutagenesis, 1800 trans-conjugants were generated and tested for tryptophan conversion. Among these, 11 mutants were selected and inoculated into wheat to compare their growth responses to the wild type. P. moraviensis wild type served as PGPR under salinity, but IAA- deficient mutants of P. moraviensis were unable to produce IAA and halted plant growth.     

Epiphytic microorganisms and IAA synthesis

Summary Epiphytic microorganisms present on cotton plants synthesized 3-indoleacetic acid (IAA) from tryptophan. Microorganisms from the root zone synthesized 3 times the amount of IAA when compared with the shoot zone and the root zone contained a much higher number of microorganisms. IAA-synthesizing activity was eliminated when the tissues were treated with a weak solution of mercuric chloride. Various tests on the possible accumulation of IAA from external sources showed that IAA synthesized outside the plant does not accumulate in the plant. Although epiphytic microorganisms synthesize IAA in large amounts, they do not influence the IAA content of the plant due to (1) lack of available tryptophan, (2) destruction of the auxin by the microflora, and (3) the polar movement of the auxin.     

Dissimilation of Tryptophan and Related Indolic Compounds by Ruminal Microorganisms In Vitro

Intraruminal doses of L-tryptophan cause acute pulmonary edema and emphysema in cattle. The D and L isomers of tryptophan and 22 related indolic compounds were incubated with ruminal microorganisms in vitro. Incubation of L-[U-benzene ring-(14)C]tryptophan with ruminal microorganisms for 24 h resulted in 39% of the added radioactivity being incorporated into skatole, 7% into indole, and 4% into indoleacetate (IAA). D-Tryptophan was not degraded to any of these metabolites. The major pathway of skatole formation from L-tryptophan appeared to be by the decarboxylation of IAA. Incubation of [2-(14)C]IAA with ruminal microorganisms for 24 h resulted in 38% incorporation into skatole. L-[5-Hydroxy]tryptophan was degraded to 5-hydroxyskatole and 5-hydroxyindole, whereas 5-hydroxyindoleacetate was degraded to only 5-hydroxyskatole. Incubation of indolepyruvate, indolelactate, and indolealdehyde with ruminal microorganisms resulted in the formation of both skatole and indole. Under similar conditions, indoleacetaldehyde was converted to IAA and tryptophol. The addition of increasing concentrations of glucose (0 to 110 mM) reduced the formation of both skatole and indole from L-tryptophan and resulted in the accumulation of IAA. Antibiotics reduced the degradation of L-tryptophan to skatole and indole, with kanamycin and neomycin particularly effective in reducing the decarboxylation of IAA to skatole.     

Catabolic Pathway for the Production of Skatole and Indoleacetic Acid by the Acetogen Clostridium drakei, Clostridium scatologenes, and Swine Manure

Skatole (3-methylindole) is a malodorous chemical in stored swine manure and is implicated as a component of foul-tasting pork. Definitive evidence for the skatole pathway is lacking. Deuterium-labeled substrates were employed to resolve this pathway in the acetogenic bacterium Clostridium drakei and Clostridium scatologenes and to determine if a similar pathway is used by microorganisms present in stored swine manure. Indoleacetic acid (IAA) was synthesized from tryptophan by both bacteria, and skatole was synthesized from both IAA and tryptophan. Microorganisms in swine manure produced skatole and other oxidation products from tryptophan, but IAA yielded only skatole. A catabolic mechanism for the synthesis of skatole is proposed.