Indole-3-pyruvic Acid Pathway Research Articles

Identification and evolution analysis of YUCCA genes of Medicago sativa and Medicago truncatula and their expression profiles under abiotic stress.

The YUCCAs (YUC) are functionally identified flavin-containing monooxidases (FMOs) in plants that act as an important rate-limiting enzyme functioning in the auxin synthesis IPA (indole-3-pyruvic acid) pathway. In this study, 12 MsYUCs and 15 MtYUCs containing characteristic conserved motifs were identified in M. sativa (Medicago sativa L.) and M. truncatula (Medicago truncatula Gaertn.), respectively. Phylogenetic analysis revealed that YUC proteins underwent an evolutionary divergence. Both tandem and segmental duplication events were presented in MsYUC and MtYUC genes. Comparative syntenic maps of M. sativa with M. truncatula, Arabidopsis (Arabidopsis thaliana), or rice (Oryza sativa L.) were constructed to illustrate the evolution relationship of the YUC gene family. A large number of cis-acting elements related to stress response and hormone regulation were revealed in the promoter sequences of MsYUCs. Expression analysis showed that MsYUCs had a tissue-specific, genotype-differential expression and a differential abiotic stress response pattern based on transcriptome data analysis of M. sativa online. In addition, RT-qPCR confirmed that salt stress significantly induced the expression of MsYUC1/MsYUC10 but significantly inhibited MsYUC2/MsYUC3 expression and the expression of MsYUC10/MsYUC11/MsYUC12 was significantly induced by cold treatment. These results could provide valuable information for functional analysis of YUC genes via gene engineering of the auxin synthetic IPA pathway in Medicago.

Complexity of the auxin biosynthetic network in Arabidopsis hypocotyls is revealed by multiple stable-labeled precursors

Auxin is a key regulator of plant development and in Arabidopsis thaliana can be synthesized through multiple pathways; however, the contributions of various biosynthetic pathways to specific developmental processes are largely unknown. To trace the involvement of various biosynthetic routes to indole-3-acetic acid (IAA) under conditions that induce adventitious root formation in Arabidopsis hypocotyls, we treated seedlings with three different stable isotope-labeled precursors ([13C6]anthranilate, [15N1]indole, and [13C3]serine) and monitored label incorporation into a number of proposed biosynthesis intermediates as well as IAA. We also employed inhibitors targeting tryptophan aminotransferases and flavin monooxygenases of the IPyA pathway, and treatment with these inhibitors differentially altered the labeling patterns from all three precursors into intermediate compounds and IAA. [13C3]Serine was used to trace utilization of tryptophan (Trp) and downstream intermediates by monitoring 13C incorporation into Trp, indole-3-pyruvic acid (IPyA), and IAA; most 13C incorporation into IAA was eliminated with inhibitor treatments, suggesting Trp-dependent IAA biosynthesis through the IPyA pathway is a dominant contributor to the auxin pool in de-etiolating hypocotyls that can be effectively blocked using chemical inhibitors. Labeling treatment with both [13C6]anthranilate and [15N1]indole simultaneously resulted in higher label incorporation into IAA through [15N1]indole than through [13C6]anthranilate; however, this trend was reversed in the proposed precursors that were monitored, with the majority of isotope label originating from [13C6]anthranilate. An even greater proportion of IAA became [15N1]-labeled compared to [13C6]-labeled in seedlings treated with IPyA pathway inhibitors, suggesting that, when the IPyA pathway is blocked, IAA biosynthesis from labeled indole may also come from an origin independent of the measured pool of Trp in these tissues.

Identification and Characterization of Auxin/IAA Biosynthesis Pathway in the Rice Blast Fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is largely unknown. By sequence homology, we identified a complete indole-3-pyruvic acid (IPA)-based IAA biosynthesis pathway in M. oryzae, consisting of the tryptophan aminotransferase (MoTam1) and the indole-3-pyruvate decarboxylase (MoIpd1). In comparison to the wild type, IAA production was significantly reduced in the motam1Δ mutant, and further reduced in the moipd1Δ mutant. Correspondingly, mycelial growth, conidiation, and pathogenicity were defective in the motam1Δ and the moipd1Δ mutants to various degrees. Targeted metabolomics analysis further confirmed the presence of a functional IPA pathway, catalyzed by MoIpd1, which contributes to IAA/auxin production in M. oryzae. Furthermore, the well-established IAA biosynthesis inhibitor, yucasin, suppressed mycelial growth, conidiation, and pathogenicity in M. oryzae. Overall, this study identified an IPA-dependent IAA synthesis pathway crucial for M. oryzae mycelial growth and pathogenic development.

Insertion of a transposon-like sequence in the 5'-flanking region of the YUCCA gene causes the stony hard phenotype.

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

Transcriptional regulation of auxin metabolic-enzyme genes during tomato fruit development

Abstract Auxin plays crucial roles in many stages of fruit development. To understand the mechanism of regulation exerted by auxin during fruit development, we analyzed the variations in the levels of indole-3-acetic acid (IAA) and its precursors, as well as the variations in the expression of the genes involved in IAA metabolism in developing tomato (Solanum lycopersicum ‘Ailsa Craig’) fruits. The concentrations of IAA and its precursor indole-3-pyruvic acid (IPyA) increased in the ovary three days after anthesis. The transcript levels of the auxin biosynthetic-enzyme genes SlTAR2, ToFZY1, ToFZY2, and ToFZY5 also increased during this stage. These results suggest that the de novo IAA biosynthesis in the pollinated ovaries is controlled through the IPyA pathway, and is involved in fruit set and early fruit growth. Contrarily, the transcripts of the auxin inactivating-enzyme genes SlGH3-9 and SlGH3-15 were accumulated in the unpollinated ovaries and decreased after pollination. This suggests that the auxin inactivating-enzymes might be involved in the control of IAA levels in the unpollinated ovaries. Further, we found that application of synthetic auxin 2,4-dichlorophenoxyacetic acid on the unpollinated ovaries increased the transcript levels of SlTAR2, ToFZY1, ToFZY2, and ToFZY5 and decreased those of SlGH3-9 and SlGH3-15. These results suggest that elevated auxin signaling via pollination or exogenous auxin treatment might promote further auxin accumulation in the ovaries.

Characterization of indole-3-pyruvic acid pathway-mediated biosynthesis of auxin in Neurospora crassa.

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 (Δcbs-3;Δahd-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 Δcbs-3;Δahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.

Re-start of olive waste vermicompost through addition of tryptophan and its effects on indole-3-acetic acid in pepper rhizosphere when used as soil amendment

In this work, we aimed to test the capacity of an exogenous source of tryptophan to stimulate the bacterial biosynthesis of the phytohormone indole-3-acetic acid (IAA) in agro-industrial by-products, trying to gain insight into the role of key genes involved in the indole-3-pyruvic acid pathway (IPA) for bacterial IAA synthesis. Sunflower-oil cake was mixed with a 9-months-old vermicompost olive wastes and then vermicomposted for 60days using earthworms Eisenia foetida. Synthetic tryptophan and no tryptophan source were used as controls. For this, a novel set of primers targeting bacterial aldehyde-dehydrogenase (ALDH)-encoding genes involved in the last step of the IPA route was designed and tested, and a method involving ultrasound-assisted extraction of IAA and subsequent analysis by liquid chromatography–tandem mass spectrometry was optimized. Re-starting the vermicomposting process increased bacterial abundance and the number and the expression of ALDH genes but not the IAA content in the final vermicomposts, meaning that the transcription of the ALDH genes could be not quite high enough to change the overall level of the existent IAA. After that, the effects of the resulting vermicompost were evaluated on the bacterial microbiome of the pepper (Capsicum annuum L.) rhizosphere. When used as amendments, ALDH gene expression was detected in the pepper’s rhizosphere after 3 months of growth only when vermicompost incorporating sunflower cake was used, where the highest levels of IAA were also detected. The results indicate the presence of the ALDH genes in the chromosome of most of vermicompost-borne bacteria, probably not specific for IAA synthesis, and that the IPA pathway may not be the main biosynthesis pathway for IAA in vermicompost-borne bacteria.

Biochemical and Chemical Biology Study of Rice OsTAR1 Revealed that Tryptophan Aminotransferase is Involved in Auxin Biosynthesis: Identification of a Potent OsTAR1 Inhibitor, Pyruvamine2031.

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

Auxin biosynthetic intermediate genes and their role in developmental growth and plasticity in higher plants

Auxins are a group of phytohormones that regulate several aspects of plant growth and development. Indole-3-acetic acid (IAA) is the predominant form of auxin in plants and several IAA biosynthetic pathways have been previously proposed but remain genetically uncharacterized. One of the proposed pathways is the indole-3-pyruvic acid (IPyA) pathway, which is inferred to regulate key developmental processes such as apical hook formation and shade avoidance. Recent molecular evidence suggests the existence of the pathway in higher plants but remains unverified due to the elusive nature of IPyA in vitro. Extending on these recent advances, this research was aimed at investigating aspects of IPyA-dependent auxin biology in Pisum sativum (pea) using reverse genetics, expression profiling, and analytical techniques. As a result the genes PsTAR2, PsTAR 5g Mt 80, and PsTAR 5g Mt 90, which are inferred to encode key enzymes in the IPyA pathway, were cloned. On expression analysis PsTAR2 was found to be slightly heightened in response to IPyA-inducing conditions (shade) while IAA levels remained unaltered contrary to previous reports. Moreover, the inferred homologs PsTAR 5g Mt 80 and PsTAR 5g Mt 90 appeared down-regulated in the same conditions suggesting functional divergence in the gene family. Thus, PsTAR2 was thought to be solely responsible for regulating IPyA-dependent auxin synthesis. Consequently, using a reverse genetic approach, called TILLING, the PsTAR2 gene was mutated in order to study the down-stream effects of IPyA deficiency. The procedure is currently underway and in the process of isolating two novel pstar2 (IPyA) mutant lines consisting of a missense mutation (pstar2 4280) and a highly desired knockout mutation (pstar2 918). On completion the novel mutants are anticipated to be indispensable to future IPyA-auxin investigations in higher plants. In light of the unstable nature of IPyA, a protocol has been formulated using UPLC for fractioning followed by MS/MS analysis. This technique appears to be very promising as a robust IPyA detection protocol in plant extracts.

Functional roles of Arabidopsis CKRC2/YUCCA8 gene and the involvement of PIF4 in the regulation of auxin biosynthesis by cytokinin.

Auxin and cytokinin (CK) are both important hormones involved in many aspects of plant growth and development. However, the details of auxin biosynthesis and the interaction between auxin and CK are still unclear. Isolation and characterization of an auxin deficient mutant cytokinin induced root curling 2 (ckrc2) in this work reveal that CKRC2 encodes a previously identified member of YUCCA (YUC) flavin monooxygenase-like proteins (YUC8). Our results show that, like other YUCs, CKRC2/YUC8 is a rate-limiting enzyme for catalyzing the conversion of indole-3-pyruvic acid (IPyA) to indole-3-acetic acid (IAA), acting downstream of CKRC1/TAA1 in the IPyA pathway. Here we show that the transcription of both CKRC1/TAA and CKRC2/YUC8 can be induced by CK and that the phytochrome-interacting factor 4 (PIF4) is required for this upregulation. Transcription of PIF4 itself is induced by CK via the AHKs-ARR1/12 signalling pathway. These results indicate that PIF4 plays an essential role in mediating the regulatory effect of CK on the transcriptions of CKRC1 and CKRC2 genes in the IPyA pathway of auxin biosynthesis.

Analysis and cloning of the synthetic pathway of the phytohormone indole-3-acetic acid in the plant-beneficial Bacillus amyloliquefaciens SQR9.

BackgroundThe 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.ResultsGene 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.ConclusionsIn conclusion, biosynthesis of IAA in B. amyloliquefaciens SQR9 occurs through multiple pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0323-4) contains supplementary material, which is available to authorized users.

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha.

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.

Transcriptional feedback regulation of YUCCA genes in response to auxin levels in Arabidopsis.

The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense.

Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2μgmg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains.

Yucasin is a potent inhibitor of YUCCA, a key enzyme in auxin biosynthesis

Indole-3-acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC-expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1-His suggested that yucasin strongly inhibited YUC1-His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over-expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l-kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin-treated sav3-2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.

Plant‐derived auxin plays an accessory role in symptom development upon Rhodococcus fascians infection

The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.

The main auxin biosynthesis pathway in Arabidopsis

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.

L-Tryptophan catabolism by Rubrivivax benzoatilyticus 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 approximately 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.

Organization of the ipdC region regulates IAA levels in different Azospirillum brasilense strains: molecular and functional analysis of ipdC in strain SM

Presence of the indole-3-pyruvic acid pathway (IPyA) of indole-3-acetic acid (IAA) biosynthesis was demonstrated by identifying the ipdC gene encoding indole-3-pyruvate decarboxylase (IPDC) in Azospirillum brasilense strain SM. Comparison with other A. brasilense strains, Sp7 and Sp245, revealed homology in the gene and its 5' regulatory region. The 3' region of strain SM carries a truncated iaaC gene implicated in controlling IAA biosynthesis in strain Sp245. While the ipdC transcript could be visualized by reverse transcription polymerase chain reaction (RT-PCR), truncated iaaC was non-functional. Strain SM derivatives carrying higher copy number of ipdC and P(ipdC) showed improved IAA biosynthesis. P(ipdC) showed sequence elements that are part of composite auxin-responsive promoters. Expression of ipdC was upregulated by IAA, other auxins, temperature and nutrient stress, and an increase in pH. Heterologous overexpression of a functional iaaC gene from strain Sp245 in strain SM confirmed its role in controlling IAA biosynthesis and lowering ipdC expression which may be effected by dissociating IAA-transcriptional regulator interactions in the 5' region. However, the effect of the introduced iaaC was overcome when both ipdC and iaaC were expressed from similar plasmid background. This analysis confirmed that strain-based differences in IAA biosynthesis could be explained by differential regulation of ipdC expression.

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.