IAA Biosynthesis Research Articles

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.

The auxin-signaling pathway is required for the lateral root response of Arabidopsis to the rhizobacterium Phyllobacterium brassicacearum

Plant root development is highly responsive both to changes in nitrate availability and beneficial microorganisms in the rhizosphere. We previously showed that Phyllobacterium brassicacearum STM196, a plant growth-promoting rhizobacteria strain isolated from rapeseed roots, alleviates the inhibition exerted by high nitrate supply on lateral root growth. Since soil-borne bacteria can produce IAA and since this plant hormone may be implicated in the high nitrate-dependent control of lateral root development, we investigated its role in the root development response of Arabidopsis thaliana to STM196. Inoculation with STM196 resulted in a 50% increase of lateral root growth in Arabidopsis wild-type seedlings. This effect was completely abolished in aux1 and axr1 mutants, altered in IAA transport and signaling, respectively, indicating that these pathways are required. The STM196 strain, however, appeared to be a very low IAA producer when compared with the high-IAA-producing Azospirillum brasilense sp245 strain and its low-IAA-producing ipdc mutant. Consistent with the hypothesis that STM196 does not release significant amounts of IAA to the host roots, inoculation with this strain failed to increase root IAA content. Inoculation with STM196 led to increased expression levels of several IAA biosynthesis genes in shoots, increased Trp concentration in shoots, and increased auxin-dependent GUS staining in the root apices of DR5::GUS transgenic plants. All together, our results suggest that STM196 inoculation triggers changes in IAA distribution and homeostasis independently from IAA release by the bacteria.

Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N2 fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C4-dicarboxylate during its symbiotic relationship with the host plant.

2. IAA-related mutants isolated by screening with IAA biosynthesis inhibitor

2. IAA-related mutants isolated by screening with IAA biosynthesis inhibitor

An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).

Indoleacetic acid concentration and metabolism changes during bud development in tubers of two potato ( Solanum tuberosum) cultivars

Plant growth regulators are involved in the control of potato (Solanum tuberosum) tuber dormancy. Evidence concerning the role of IAA is controversial; we therefore investigated its role by analyzing two cultivars with varying lengths of dormancy. We examined the time course of free and conjugated IAA in tuber tissue isolates from the final stages of tuber growth to the end of dormancy, the distribution of free IAA in tuber tissues by in situ analysis, and the biosynthesis of the hormone by feeding experiments. The time course of free IAA showed marked differences between the examined cultivars, although the concentration of the auxin generally was the highest at the early stages of tuber dormancy. Immunodetection showed a similar pattern of IAA distribution in both genotypes: in dormant buds from freshly harvested tubers, the free hormone accumulated mostly in apical meristem, leaf and lateral bud primordia, and differentiating vascular tissues underlying the apical meristem, while at the end of the storage period only axillary bud primordia from growing buds displayed appreciable auxin levels. Feeding experiments indicated that changes in IAA biosynthesis rate were a major cause of auxin variation in buds. In both cultivars, dormancy apparently ceased when free IAA fell below a threshold value. Despite this, our data led us to conclude that IAA would not be directly responsible for inhibiting sprouting. Instead, auxin might shorten dormancy, in a cultivar-dependent manner, by enhancing early developmental processes in buds, ultimately leading to dormancy termination.

10. Is the tissue/cell structure of coleoptile tip necessary for IAA biosynthesis in maize coleoptiles?

10. Is the tissue/cell structure of coleoptile tip necessary for IAA biosynthesis in maize coleoptiles?

5. IAA mutants isolated by screening with IAA biosynthesis inhibitor

5. IAA mutants isolated by screening with IAA biosynthesis inhibitor

Glucosinolates and the clubroot disease: defense compounds or auxin precursors?

The clubroot disease is caused by the obligate biotrophic protist Plasmodiophora brassicae and is one of the most damaging for the family of Brassicaceae. Since many economically important crops belong to this plant family, the understanding of mechanisms how the disease is developing, are of high importance. Glucosinolates, a group of secondary plant products in the family of Brassicaceae, have long been associated with clubroot disease symptoms. Measurements showed that several glucosinolates are induced in root galls. While aliphatic glucosinolates are regarded as defense compounds, analysis of Brassica cultivars as well as Arabidopsis thaliana mutants provided correlative evidence between disease severity and indole glucosinolate content. The latter have been discussed as precursors for auxin biosynthesis. Since high auxin levels are associated with large root galls, indole glucosinolates could contribute directly or indirectly to the extent of disease development. Transcriptome and proteome experiments have revealed evidence for the involvement of genes from the glucosinolate and auxin pathway in gall formation. These data have been complemented by expression and mutant analysis. It can be concluded that regulation of glucosinolate and IAA biosynthesis might differ in Brassica and Arabidopsis.

An ipdC gene knock-out of Azospirillum brasilense 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 approximately 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.

8. IAA biosynthesis at the tip region and its transport in maize coleoptiles

8. IAA biosynthesis at the tip region and its transport in maize coleoptiles

Moss-associated methylobacteria as phytosymbionts: an experimental study

Methylotrophic bacteria inhabit the surface of plant organs, but the interaction between these microbes and their host cells is largely unknown. Protonemata (gametophytes) of the moss Funaria hygrometrica were cultivated in vitro under axenic conditions and the growth of the protonemal filaments recorded. In the presence of methylobacteria (different strains of Methylobacterium), average cell length and the number of cells per filament were both enhanced. We tested the hypothesis that auxin (indole-3-acetic acid, IAA), secreted by the epiphytic bacteria and taken up by the plant cells, may in part be responsible for this promotion of protonema development. The antiauxin parachlorophenoxyisobutyric acid (PCIB) was used as a tool to analyze the role of IAA and methylobacteria in the regulation of cell growth. In the presence of PCIB, cell elongation and protonema differentiation were both inhibited. This effect was compensated for by the addition of different Methylobacterium strains to the culture medium. Biosynthesis and secretion of IAA by methylobacteria maintained in liquid culture was documented via a colorimetric assay and thin layer chromatography. Our results support the hypothesis that the development of Funaria protonemata is promoted by beneficial phytohormone-producing methylobacteria, which can be classified as phytosymbionts.

Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles

When maize coleoptiles were unilaterally exposed to red light (7.9 micromol m(-2)s(-1) for 5 min), 3 h after treatment IAA levels in coleoptiles decreased in all regions, from top to basal, with levels about 60% of dark controls. Localized irradiation in the 5 mm top zone was sufficient to cause the same extent of IAA reduction in the tips to that in the tips of whole irradiated shoots. When coleoptiles were treated with N-1-naphthylphthalamic acid (NPA), an accumulation of IAA in the tip and a decrease of diffusible IAA from tips were simultaneously detected. IAA accumulation in red-light treated coleoptiles by NPA was much lower than that of dark controls. NPA treatment did not affect the content of conjugated IAA in either dark or light treated coleoptile tips. When (13)C(11) (15)N(2)-tryptophan (Trp) was applied to the top of coleoptiles, substantial amounts of stable isotope were incorporated into free IAA in dark and red-light treated coleoptile tips. The ratio of incorporation was slightly lower in red-light treated coleoptile tips than that in dark controls. The label could not be detected in conjugated IAA. The rate of basipetal transport of IAA was about 10 mm h(-1) and the velocity was not affected by red light. These results strongly suggest that red light does not affect the rates of conversion of free IAA to the conjugate form or of the basipetal transport, but just reduces the IAA level in the tips, probably inhibited by IAA biosynthesis from Trp in this region.

Hrp-dependent biotrophic mechanism of virulence: How has it evolved in tumorigenic bacteria?

A mechanism of virulence mediated byhrp-genes is present in many Gram-negative bacterial pathogens. It involves delivery of effector proteins into host cellsvia the type III secretion system (TTSS) and the interaction of TTSS effectors with plant proteins. These interactions may either promote responses beneficial to the pathogen or trigger the hypersensitive response if an effector is recognized by corresponding resistance protein.Pantoea agglomerans, which is widespread in nature mainly as an epiphyte, has evolved into ahrp-dependent and host-specific tumorigenic pathogen by acquiring a plasmid containing a pathogenicity island (PAI). This PAI harbors ahrp-gene cluster, and genes encoding for TTSS effector proteins and biosynthesis of IAA and cytokinins. The results reviewed describe how the interplay between negative-acting and positive-acting TTSS effectors determines the transformation ofP. agglomerans into two related pathovars. Furthermore, the PAI’s structure supports the premise that these pathovars are recently evolved pathogens. Finally, the possible interaction between TTSS effectors and phytohormones for gall formation is proposed.

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 °C in a standard minimal medium (MMAB) containing 2.5 g l −1 l-malate and 50 μg ml −1 tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene ( ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.

Perrot-Rechenmann, C. and Hagen, G. Auxin molecular biology

Perrot-Rechenmann, C. and Hagen, G. Auxin molecular biology

An auxin surge following fertilization in carrots: a mechanism for regulating plant totipotency.

All plants exhibit the property of cellular totipotency, whereby individual cells can regenerate into an entire organism. However little is known about the underlying mechanisms regulating totipotency. Using a preparative microtechnique, we report an 80-fold surge in the concentration of free auxin that is correlated with the initial stages of zygotic embryogenesis in carrots. The concentration of free IAA increases from a basal level of ca. 25 ng/g FW in unfertilized ovules to ca. 2.000 ng/g FW in the late globular and early heart stages, then back to the basal level in the torpedo stage. This initial increase in IAA levels is diagnostic of the activity of the tryptophan-mediated pathway for IAA biosynthesis, while the maintenance of the basal levels is attributed to the tryptophan-independent pathway for IAA biosynthesis. Our observations are consistent with the hypothesis that the sequential activation of alternative IAA biosynthetic pathways is a critical mechanism for regulating carrot (Daucus carota L. cv. Danvers 126) embryogenesis and other instances of plant totipotency.

Indole-3-acetic acid biosynthesis in isolated axes from germinating bean seeds: The effect of wounding on the biosynthetic pathway

A facile radiotracer method for discriminating between the activities of tryptophan-dependent and tryptophanindependent pathways for IAA biosynthesis is described. This method utilized the simultaneous exposure of plants to [ 14 C]anthranilate in the presence or absence of excess unlabeled tryptophan in order to determine if tryptophan feeding can affect the relative enrichment of the IAA pool. Using this radiotracer method, the activities of the two biosynthetic pathways were analyzed in isolated axes of germinating bean seedlings at various times after cotyledon excision. Unlabeled tryptophan suppressed [ 14 C]anthranilate conversion into IAA in isolated axes of different ages immediately following cotyledon excision. On the other hand, tryptophan feeding did not inhibit [ 14 C]IAA accumulation in isolated axes 36 or 120 h after cotyledon excision. Thus, this method was able to resolve time-dependent differences following cotyledon excision in the biosynthetic activities of the two pathways. Moreover, the present results lend further support to the emerging consensus that the tryptophan-dependent pathway acts to maintain very high IAA levels required for mediating rapid cell proliferation in wounded tissues and, as previously shown, young embryos.

Evolutionary patterns in auxin action.

This review represents the first effort ever to survey the entire literature on auxin (indole-3-acetic acid, IAA) action in all plants, with special emphasis on the green plant lineage, including charophytes (the green alga group closest to the land plants), bryophytes (the most basal land plants), pteridophytes (vascular non-seed plants), and seed plants. What emerges from this survey is the surprising perspective that the physiological mechanisms for regulating IAA levels and many IAA-mediated responses found in seed plants are also present in charophytes and bryophytes, at least in nascent forms. For example, the available evidence suggests that the apical regions of both charophytes and liverworts synthesize IAA via a tryptophan-independent pathway, with IAA levels being regulated via the balance between the rates of IAA biosynthesis and IAA degradation. The apical regions of all the other land plants utilize the same class of biosynthetic pathway, but they have the potential to utilize IAA conjugation and conjugate hydrolysis reactions to achieve more precise spatial and temporal control of IAA levels. The thallus tips of charophytes exhibit saturable IAA influx and efflux carriers, which are apparently not sensitive to polar IAA transport inhibitors. By contrast, two divisions of bryophyte gametophytes and moss sporophytes are reported to carry out polar IAA transport, but these groups exhibit differing sensitivities to those inhibitors. Although the IAA regulation of charophyte development has received almost no research attention, the bryophytes manifest a wide range of developmental responses, including tropisms, apical dominance, and rhizoid initiation, which are subject to IAA regulation that resembles the hormonal control over corresponding responses in seed plants. In pteridophytes, IAA regulates root initiation and vascular tissue differentiation in a manner also very similar to its effects on those processes in seed plants. Thus, it is concluded that the seed plants did not evolve de novo mechanisms for mediating IAA responses, but have rather modified pre-existing mechanisms already operating in the early land plants. Finally, this paper discusses the encouraging prospects for investigating the molecular evolution of auxin action.

Mutual interaction of auxin and cytokinins in regulating correlative dominance

Correlative dominance requires correlative signals from a dominant to a dominated organ. Auxins, particularly IAA, and cytokinins are obviously important components of this correlative system. Using a vegetative pea shoot and a generative apple and tomato fruit system it can be demonstrated that dominant organs always export more IAA and have a higher 3H-IAA transport capacity and velocity compared to dominated organs. In both systems the dominant organ can be replaced by the application of auxin, e.g. NAA, which maintains the differences in IAA export. This is an indication that similar regulatory mechanisms control dominance in both of these diverse systems. The possibility of replacing a dominant organ by auxin also makes it unlikely that growth of that organ or allocation of nutrients regulates the correlative inhibition of the dominated organ.It is suggested that differences in IAA export from, and transport capacities of, dominant and dominated shoots, may be explained by a mechanism of auxin transport autoinhibition (ATA), whereby the earlier and stronger export of IAA from the dominant shoot inhibits auxin export from the dominated shoot at the point where the two auxin streams converge. This hypothesis was tested with explants of pea, apple and tomato. It was shown that the basal application of cold IAA significantly reduced endogenous as well as exogenous IAA transport through these explants.Since the reduced IAA transport of dominated organs was not followed by an accumulation of IAA in the auxin producing subtending organ, it was concluded that IAA biosynthesis was possibly reduced and/or IAA conjugation stimulated. This could have been one of the determinants of their growth inhibition. ATA might also explain how the unidirectional IAA signal may affect the growth rate of organs even lateral or acropetal to its transport pathway and thus polar IAA-transport becomes a ``multidirectional'' signal. From the experiments demonstrated it seems that ATA is a sufficient mechanism to impose growth inhibition in the dominated organ, without the need of other regulators.However, to release dominated organs from dominance cessation of ATA may not be sufficient and cytokinins are obviously a powerful antagonist to auxins. Their repeated exogenous application turns dominated lateral buds into strongly growing organs which ultimately may even dominate the previously dominant apex. These lateral shoots finally gain a strong IAA export capacity and inhibit, by ATA, IAA export from the hitherto dominant apex.In other experiments it was shown that interruption of polar IAA transport leads to a strong increase in root derived cytokinins. This can largely be prevented, in a concentration dependent manner, by the application of auxin, indicating that basipolar auxin may control cytokinin production in the roots and its possible delivery to lateral buds. In turn, the increased delivery of cytokinins to the lateral buds promotes a strong increase in IAA production and export. Thus there is a strong mutual interaction between auxin production in the shoots and cytokinin production in the roots, which may be important in regulating the balance between root and shoot growth.