Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples.
ABSTRACT: BACKGROUND: The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis. RESULTS: We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2-10?mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods. CONCLUSIONS: The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA.
Project description:Auxins are hormones that regulate many aspects of plant growth and development. The main plant auxin is indole-3-acetic acid (IAA), whose biosynthetic pathway is not fully understood. Indole-3-acetaldoxime (IAOx) has been proposed to be a key intermediate in the synthesis of IAA and several other indolic compounds. Genetic studies of IAA biosynthesis in Arabidopsis have suggested that 2 distinct pathways involving the CYP79B or YUCCA (YUC) genes may contribute to IAOx synthesis and that several pathways are also involved in the conversion of IAOx to IAA. Here we report the biochemical dissection of IAOx biosynthesis and metabolism in plants by analyzing IAA biosynthesis intermediates. We demonstrated that the majority of IAOx is produced by CYP79B genes in Arabidopsis because IAOx production was abolished in CYP79B-deficient mutants. IAOx was not detected from rice, maize, and tobacco, which do not have apparent CYP79B orthologues. IAOx levels were not significantly altered in the yuc1 yuc2 yuc4 yuc6 quadruple mutants, suggesting that the YUC gene family probably does not contribute to IAOx synthesis. We determined the pathway for conversion of IAOx to IAA by identifying 2 likely intermediates, indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN), in Arabidopsis. When (13)C(6)-labeled IAOx was fed to CYP79B-deficient mutants, (13)C(6) atoms were efficiently incorporated to IAM, IAN, and IAA. This biochemical evidence indicates that IAOx-dependent IAA biosynthesis, which involves IAM and IAN as intermediates, is not a common but a species-specific pathway in plants; thus IAA biosynthesis may differ among plant species.
Project description: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.
Project description:<h4>Background</h4>The plant hormone auxin plays a central role in regulation of plant growth and response to environmental stimuli. Multiple pathways have been proposed for biosynthesis of indole-3-acetic acid (IAA), the primary auxin in a number of plant species. However, utilization of these different pathways under various environmental conditions and developmental time points remains largely unknown.<h4>Results</h4>Monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates provides a method to trace pathway utilization and characterize new biosynthetic routes to auxin. These techniques can be aided by addition of chemical inhibitors to target specific steps or entire pathways of auxin synthesis.<h4>Conclusions</h4>Here we describe techniques for pathway analysis in Arabidopsis thaliana seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry (LC-MS) methods. These methods should prove to be useful to researchers studying routes of IAA biosynthesis in vivo in a variety of plant tissues.
Project description:The plant hormone auxin regulates many aspects of plant growth and development. Although several auxin biosynthetic pathways have been proposed, none of these pathways has been precisely defined at the molecular level. Here we provide in planta evidence that the two Arabidopsis cytochrome P450s, CYP79B2 and CYP79B3, which convert tryptophan (Trp) to indole-3-acetaldoxime (IAOx) in vitro, are critical enzymes in auxin biosynthesis in vivo. IAOx is thus implicated as an important intermediate in auxin biosynthesis. Plants overexpressing CYP79B2 contain elevated levels of free auxin and display auxin overproduction phenotypes. Conversely, cyp79B2 cyp79B3 double mutants have reduced levels of IAA and show growth defects consistent with partial auxin deficiency. Together with previous work on YUCCA, a flavin monooxygenase also implicated in IAOx production, and nitrilases that convert indole-3-acetonitrile to auxin, this work provides a framework for further dissecting auxin biosynthetic pathways and their regulation.
Project description:The majority of clinically used antibiotics originate from bacteria. As the need for new antibiotics grows, large-scale genome sequencing and mining approaches are being used to identify novel antibiotics. However, this task is hampered by the fact that many antibiotic biosynthetic clusters are not expressed under laboratory conditions. One strategy to overcome this limitation is the identification of signals that activate the expression of silent biosynthetic pathways. Here, we report the use of high-throughput screening to identify signals that control the biosynthesis of the acetyl-CoA carboxylase inhibitor antibiotic andrimid in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. We reveal that the pathway-specific transcriptional activator AdmX recognizes the auxin indole-3-acetic acid (IAA). IAA binding causes conformational changes in AdmX that result in the inhibition of the expression of the andrimid cluster and the suppression of antibiotic production. We also show that IAA synthesis by pathogenic and beneficial plant-associated bacteria inhibits andrimid production in A153. Because IAA is a signalling molecule that is present across all domains of life, this study highlights the importance of intra- and inter-kingdom signalling in the regulation of antibiotic synthesis. Our discovery unravels, for the first time, an IAA-dependent molecular mechanism for the regulation of antibiotic synthesis.
Project description:<h4>Background</h4>The plant hormone auxin is a major coordinator of plant growth and development in response to diverse environmental signals, including nutritional conditions. Sole ammonium (NH<sub>4</sub><sup>+</sup>) nutrition is one of the unique growth-suppressing conditions for plants. Therefore, the quest to understand NH<sub>4</sub><sup>+</sup>-mediated developmental defects led us to analyze auxin metabolism.<h4>Results</h4>Indole-3-acetic acid (IAA), the most predominant natural auxin, accumulates in the leaves and roots of mature Arabidopsis thaliana plants grown on NH<sub>4</sub><sup>+</sup>, but not in the root tips. We found changes at the expressional level in reactions leading to IAA biosynthesis and deactivation in different tissues. Finally, NH<sub>4</sub><sup>+</sup> nutrition would facilitate the formation of inactive oxidized IAA as the final product.<h4>Conclusions</h4>NH<sub>4</sub><sup>+</sup>-mediated accelerated auxin turnover rates implicate transient and local IAA peaks. A noticeable auxin pattern in tissues correlates with the developmental adaptations of the short and highly branched root system of NH<sub>4</sub><sup>+</sup>-grown plants. Therefore, the spatiotemporal distribution of auxin might be a root-shaping signal specific to adjust to NH<sub>4</sub><sup>+</sup>-stress conditions.
Project description: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 (?cbs-3;?ahd-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 ?cbs-3;?ahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.
Project description:To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.
Project description:The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.
Project description:Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.