Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

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.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.     

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.     

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.     

Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation

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.     

Functional identification and expression of indole-3-pyruvate decarboxylase from Paenibacillus polymyxa E681

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylaselike proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5alpha. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.     

Indole-3-acetic acid in plant–microbe interactions

Indole-3-acetic acid (IAA) is an important phytohormone with the capacity to control plant development in both beneficial and deleterious ways. The ability to synthesize IAA is an attribute that many bacteria including both plant growth-promoters and phytopathogens possess. There are three main pathways through which IAA is synthesized; the indole-3-pyruvic acid, indole-3-acetamide and indole-3-acetonitrile pathways. This chapter reviews the factors that effect the production of this phytohormone, the role of IAA in bacterial physiology and in plant–microbe interactions including phytostimulation and phytopathogenesis.     

Conversion of D-tryptophan to indole-3-acetic acid in coleoptiles of a normal and a semi-dwarf barley (Hordeum vulgare) strain

Exogenously applied D-tryptophan (D-Trp) was more effective than L-Trp in inducing elongation of coleoptile segments of a normal barley ( Hordeum vulgare L. cv. Akashinriki) strain and a semi-dwarf strain with lower endogenous indole-3-acetic acid (IAA) level. D-cycloserine, an inhibitor of D-aminotransferase, completely inhibited both the D- and L-Trp-induced elongation of both strains. Addition of D-Trp increased IAA levels in both strains 4-fold over endogenous levels. The increase in IAA level was completely inhibited by D-cycloserine. The endogenous L-Trp level of semi-dwarf coleoptiles was similar to that of normal ones. These results suggested that IAA is synthesized by the conversion of L-Trp to indole-3-pyruvic acid via D-Trp in both strains, and that the lower IAA level of the semi-dwarf strain probably is a result of the impeded IAA biosynthesis involved in D-Trp.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan catabolism by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2 occurs through indole 3-pyruvic acid pathway

Rubrivivax benzoatilyticus JA2 utilizes l-tryptophan as the sole source of nitrogen for growth, and it has a doubling time of ~11 h (compared to 8 h with ammonium chloride). With cell free extracts in the presence of 2-oxoglutarate, indole-3-pyruvic acid, indole-3-acetaldehyde, indole-3-acetic acid, isatin, benzaldehyde, gallic acid and pyrogallol were identified using high performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC–MS) analysis. The conversion of l-tryptophan into indole 3-pyruvic acid and glutamate by an enzyme aminotransferase was confirmed and the catabolism of indole-3-pyruvic acid via side chain oxidation followed by ring oxidation, gallic acid and pyrogallol were confirmed as metabolites. In addition, the proposed pathway sequential conversion of indole-3-pyruvic acid to the end product of pyrogallol was identified, including an enzymatic step that would convert isatin to benzaldehyde by an enzyme yet to be identified. At this stage of the study, the enzyme tryptophan aminotransferase in R. benzoatilyticus JA2 was demonstrated.     

An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading grame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.     

Increased urinary excretion of aromatic amino acid catabolites by Microtus montanus chronically infected with Trypanosoma brucei gambiense

Microtus montanus infected with Trypanosoma brucei gambiense for 16 and 21 days excreted significantly greater quantities of several aromatic amino acid catabolites when compared to uninfected control animals. Very large quantities of three aromatic alpha-keto acids (alpha-oxocarboxylic acids), phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid, were excreted by infected animals. Increased excretion of indole-3-lactic acid and indole-3-acetic acid was also detected. Gas chromatographic-mass spectral analysis of the trimethylsilyl derivatives of phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid confirms the identity of the aromatic alpha-keto acids elevated during infection. The marked alpha-keto aciduria indicates that a large disturbance exists in aromatic amino acid metabolism in this chronic animal model of African trypanosomiasis. The disturbance may contribute to the pathogenesis of the disease. The increased catabolite concentrations may also prove to be useful diagnostically and prognostically.     

Comparison of different enzyme immunoassays for measuring indole-3-acetic acid in vegetative citrus tissues

An enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies, which were raised against indole-3-acetic acid (IAA) conjugated to bovine serum albumin (BSA) via the indolic nitrogen (IAA-N₁-BSA), has been developed. The sensitivity and specificity of these antibodies were compared to those of polyclonal and monoclonal antibodies raised against IAA conjugated to BSA via C₁ of the carboxyl group (IAA-C₁-BSA). The sensitivity of the assays improved in the following order: monoclonal antibodies > antibodies to IAA-C₁-BSA > antibodies to IAA-C₁-BSA. Antibodies against IAA-C₁-BSA had less cross-reactivity to indoles structurally related to IAA, excluding indole-3-pyruvic acid. A rapid and effective method for purification of IAA in citrus tissues before analysis by ELISA is described. Values of IAA in citrus ( Citrus sinensis [L.] Osbeck cv. Shamouti orange) shoot tips obtained with all three antibodies were similar. However, in leaf tissues which contain lower amounts of IAA compared to shoot tips, monoclonal antibodies gave higher values of IAA than polyclonal antibodies. Estimation of free IAA levels in purified extracts of citrus shoot tips, very young leaves, and mature leaves was ca 380, 248, and 74 ng (g fresh weight)⁻¹ respectively.     

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid

Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant–microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.