Indole-3-acetic Acid Biosynthesis Research Articles

Auxin metabolism and function in the multicellular brown alga Ectocarpus siliculosus.

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.

Modulation of IAA Production in Cyanobacteria by Tryptophan and Light

Cyanobacteria represent less a investigated group of prokaryote, in terms of their plant growth promoting potential, especially in relation to the production of phytohormones. The present investigation was aimed towards analyzing growth kinetics, indole acetic acid (IAA) production and acetylene reduction activity (ARA) as an index of nitrogen fixation in two selected cyanobacterial strains belonging to the genus Anabaena, as influenced by tryptophan supplementation and light:dark conditions. Interesting observations were recorded in terms of enhancement of IAA production accompanied by protein and chlorophyll accumulation in the two cyanobacterial strains grown in media without tryptophan and incubated under light:dark or continuous light conditions. Colorimetric and chromatographic analyses supported the observations that tryptophan is not essential as a precursor for IAA biosynthesis in these cyanobacteria. Further study is in progress to identify genes involved in the tryptophan independent pathway for IAA biosynthesis.

Approaching Cellular and Molecular Resolution of Auxin Biosynthesis and Metabolism

There is abundant evidence of multiple biosynthesis pathways for the major naturally occurring auxin in plants, indole-3-acetic acid (IAA), and examples of differential use of two general routes of IAA synthesis, namely Trp-dependent and Trp-independent. Although none of these pathways has been completely defined, we now have examples of specific IAA biosynthetic pathways playing a role in developmental processes by way of localized IAA synthesis, causing us to rethink the interactions between IAA synthesis, transport, and signaling. Recent work also points to some IAA biosynthesis pathways being specific to families within the plant kingdom, whereas others appear to be more ubiquitous. An important advance within the past 5 years is our ability to monitor IAA biosynthesis and metabolism at increasingly higher resolution.

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 (tdd1), which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase beta-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1, indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1, which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR, which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.

Regulatory Interactions Between Quorum-Sensing, Auxin, Cytokinin, and the Hrp Regulon in Relation to Gall Formation and Epiphytic Fitness of Pantoea agglomerans pv. gypsophilae

Gall formation by Pantoea agglomerans pv. gypsophilae is controlled by hrp/hrc genes, phytohormones, and the quorum-sensing (QS) regulatory system. The interactions between these three components were investigated. Disruption of the QS genes pagI and pagR and deletion of both substantially reduced the transcription levels of the hrp regulatory genes hrpXY, hrpS, and hrpL, as determined by quantitative reverse-transcriptase polymerase chain reaction. Expression of hrpL in planta was inhibited by addition of 20 microM or higher concentrations of the QS signal C(4)-HSL. The pagR and hrpL mutants caused an equivalent reduction of 1.3 orders in bacterial multiplication on bean leaves, suggesting possible mediation of the QS effect on epiphytic fitness of P. agglomerans pv. gypsophilae by the hrp regulatory system. indole-3-acetic acid (IAA) and cytokinin significantly affected the expression of the QS and hrp regulatory genes. Transcription of pagI, pagR, hrpL, and hrpS in planta was substantially reduced in iaaH mutant (disrupted in IAA biosynthesis via the indole-3-acetamide pathway) and etz mutant (disrupted in cytokinin biosynthesis). In contrast, the ipdC mutant (disrupted in IAA biosynthesis via the indole-3-pyruvate pathway) substantially increased expression of pagI, pagR, hrpL, and hrpS. Results presented suggest the involvement of IAA and cytokinins in regulation of the QS system and hrp regulatory genes.

Effects of indole-3-acetic acid on Sinorhizobium meliloti survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and alpha-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-beta-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.

Plant growth promoting potential of the fungus Discosia sp. FIHB 571 from tea rhizosphere tested on chickpea, maize and pea.

The ITS region sequence of a phosphate-solubilizing fungus isolated from the rhizosphere of tea growing in Kangra valley of Himachal Pradesh showed 96% identity with Discosia sp. strain HKUCC 6626 ITS 1, 5.8S rRNA gene and ITS 2 complete sequence, and 28S rRNA gene partial sequence. The fungus exhibited the multiple plant growth promoting attributes of solubilization of inorganic phosphate substrates, production of phytase and siderophores, and biosynthesis of indole acetic acid (IAA)-like auxins. The fungal inoculum significantly increased the root length, shoot length and dry matter in the test plants of maize, pea and chickpea over the uninoculated control under the controlled environment. The plant growth promoting attributes have not been previously studied for the fungus. The fungal strain with its multiple plant growth promoting activities appears attractive towards the development of microbial inoculants.

Controlled indole-3-acetaldoxime production through ethanol-induced expression of CYP79B2

Indole-3-acetaldoxime (IAOx) is a key branching point between primary and secondary metabolism. IAOx serves as an intermediate in the biosynthesis of indole glucosinolates (I-GLSs), camalexin and the plant hormone indole-3-acetic acid (IAA). The cytochrome P450s CYP79B2 and CYP79B3 catalyze the conversion of tryptophan to IAOx. CYP83B1 channels IAOx into I-GLS biosynthesis, CYP71A13 channels IAOx into camalexin biosynthesis, whereas the IAOx-metabolizing enzyme in IAA biosynthesis is not known. In this report, we demonstrate controlled production of I-GLSs by introducing an ethanol (EtOH)-inducible CYP79B2 construct into double (cyp79b2 cyp79b3) or triple (cyp79b2 cyp79b3 cyp83b1) mutant lines. We show EtOH-dependent induction of camalexin and identify a number of candidate IAA homeostasis- or defense-related genes by clustered microarray analysis. The transgenic mutant lines are thus promising tools for elucidating the interplay between primary and secondary metabolism.

Production of the Phytohormone Indole-3-Acetic Acid by Estuarine Species of the Genus Vibrio

Strains of Vibrio spp. isolated from roots of the estuarine grasses Spartina alterniflora and Juncus roemerianus produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA, recA, and rpoA genes was used for phylogenetic analysis of environmental isolates within the genus Vibrio. Eight Vibrio type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing Vibrio species. Six additional environmental isolates similar to four of the Vibrio type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment.

Arabidopsis Indole Synthase, a Homolog of Tryptophan Synthase Alpha, is an Enzyme Involved in the Trp‐independent Indole‐containing Metabolite Biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions. Indole-3-acetic acid (IAA), proposed as a derivative from Trp or its precursors, plays an essential role in plant growth and development. Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed, the enzymes, reactions and regulatory mechanisms are largely unknown. In Arabidopsis, indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis. To address whether other enzymes in addition to Trp synthase alpha (TSA1) catalyze IGP cleavage, we identified and characterized an indole synthase (INS) gene, a homolog of TSA1 in Arabidopsis. INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP). In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different. Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons, hypocotyls, roots and rosette leaves as well as in flowers and siliques. INS is capable of complementing the Trp auxotrophy of Escherichia coliDeltatrpA strain, which is defective in Trp synthesis due to the deletion of TSA. This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.

Potential pathways of indole acetic acid (IAA) biosynthesis in Euphorbia abyssinica

AbstractDesert candle (Euphorbia abyssinica J.F. Gmel.) (further referred to as EAG) is a succulent tree of dry deciduous and evergreen montane forest, woodland and shrub savanna. It occurs widely throughout dryland Africa, where it is appreciated as a live fence because it is easily propagated from untreated mature branch cuttings.Earlier we argued that the ability of large EAG branches to regenerate with ease in dry soil may be related to the natural plant growth regulator hormone indole acetic acid (IAA) contained in the latex of the plant. EAG latex samples from northern Ethiopia (n = 3) that were chemically analyzed contained on average 0.06 mg/l latex, as well as the IAA metabolites indole lactate (ILA) and indole ethanol (IEt). One sample also contained IAA conjugated to amino-acids and to glucose. Here we present a schematic overview of the potential pathways of IAA biosynthesis in EAG and suggest that auxin biosynthesis in EAG is tryptophane (Trp) dependent.

Potential pathways of indole acetic acid (IAA) biosynthesis in Euphorbia abyssinica

Desert candle (Euphorbia abyssinica J.F. Gmel.) (further referred to as EAG) is a succulent tree of dry deciduous and evergreen montane forest, woodland and shrub savanna. It occurs widely throughout dryland Africa, where it is appreciated as a live fence because it is easily propagated from untreated mature branch cuttings.Earlier we argued that the ability of large EAG branches to regenerate with ease in dry soil may be related to the natural plant growth regulator hormone indole acetic acid (IAA) contained in the latex of the plant. EAG latex samples from northern Ethiopia (n = 3) that were chemically analyzed contained on average 0.06 mg/l latex, as well as the IAA metabolites indole lactate (ILA) and indole ethanol (IEt). One sample also contained IAA conjugated to amino-acids and to glucose. Here we present a schematic overview of the potential pathways of IAA biosynthesis in EAG and suggest that auxin biosynthesis in EAG is tryptophane (Trp) dependent.

The Role of Aspterric Acid in Auxin-Regulated Reproductive Growth of Arabidopsis thaliana

Application of 100 microM aspterric acid (AA), a pollen growth inhibitor, with different concentrations of indole-3-acetic acid (IAA) results in the recovery of normal pollen development of Arabidopsis thaliana. Treatment with 100 microM AA plus 5 mM IAA significantly induced the normal seed production. Treatment with 100 microM N-1-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor, did not reduce the pollen growth but inhibited seed production. 100 microM NPA plus 5 mM IAA did not induce any seed production. The endogenous level of IAA in stems and leaves of A. thaliana treated with 100 microM AA was similar to that of the untreated control. In contrast to AA treatment, the IAA level by the treatment with 100 microM NPA was about twice as much as that of the untreated control. These results suggest that AA affects the Arabidopsis reproductive growth without inhibiting IAA biosynthesis and transport.

Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.

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.

A New Gene for Auxin Synthesis

There is much interest in understanding the pathways that trigger biosynthesis of the plant hormone auxin. In this issue, Stepanova et al. (2008) and Tao et al. (2008) reveal that a small family of tryptophan aminotransferases catalyze formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a pathway for auxin biosynthesis.

Isolation and Characterization of Transposon-Insertional Mutants from Paenibacillus polymyxa E681 Altering the Biosynthesis of Indole-3-Acetic Acid

We screened a mini-Tn10 insertional mutant library of the spore-forming bacterium Paenibacillus polymyxa E681 with variable indole-3-acetic acid (IAA) productivity. Four mutants, of which two showed a decrease in IAA production and the other two showed an increase in IAA production, were finally selected. Further analyses demonstrated different levels of IAA intermediates from culture supernatant of wild-type strain and mutants. In addition, mutants showed different promotions on the early growth of 10-day-old maize in terms of the increase in shoot and root weights. DNA fragments flanking the transposon insertion in four mutants were cloned and sequenced. The target sites of insertion were gene gpr1, disrupted at two sites, 49 bp downstream of the spo0F gene, and relA/spoT homologue, which codes for GPR1/FUN34/YaaH family protein, stage 0 sporulation protein F, and RelA/SpoT domain protein, respectively. This evidence suggests that there may be a number of genes involved in the regulation of IAA biosynthesis of P. polymyxa.

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.

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.

74. Search for new small molecules which affect IAA (indole-3-acetic acid) biosynthesis in maize coleoptile tips by a 10000-chemical library

74. Search for new small molecules which affect IAA (indole-3-acetic acid) biosynthesis in maize coleoptile tips by a 10000-chemical library