Biomedical subjects

Björn Usadel

Publications and source records attributed to Björn Usadel.

13 recordsLinked to original sources

PubMed2007

The lipopolysaccharide of Sinorhizobium meliloti suppresses defense-associated gene expression in cell cultures of the host plant Medicago truncatula.

In the establishment of symbiosis between Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti, the lipopolysaccharide (LPS) of the microsymbiont plays an important role as a signal molecule. It has been shown in cell cultures that the LPS is able to suppress an elicitor-induced oxidative burst. To investigate the effect of S. meliloti LPS on defense-associated gene expression, a microarray experiment was performed. For evaluation of the M. truncatula microarray datasets, the software tool MapMan, which was initially developed for the visualization of Arabidopsis (Arabidopsis thaliana) datasets, was adapted by assigning Medicago genes to the ontology originally created for Arabidopsis. This allowed functional visualization of gene expression of M. truncatula suspension-cultured cells treated with invertase as an elicitor. A gene expression pattern characteristic of a defense response was observed. Concomitant treatment of M. truncatula suspension-cultured cells with invertase and S. meliloti LPS leads to a lower level of induction of defense-associated genes compared to induction rates in cells treated with invertase alone. This suppression of defense-associated transcriptional rearrangement affects genes induced as well as repressed by elicitation and acts on transcripts connected to virtually all kinds of cellular processes. This indicates that LPS of the symbiont not only suppresses fast defense responses as the oxidative burst, but also exerts long-term influences, including transcriptional adjustment to pathogen attack. These data indicate a role for LPS during infection of the plant by its symbiotic partner.

Cells, Cultured↗

PubMed2007

Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus.

Affymetrix ATH1 arrays, large-scale real-time reverse transcription PCR of approximately 2200 transcription factor genes and other gene families, and analyses of metabolites and enzyme activities were used to investigate the response of Arabidopsis to phosphate (Pi) deprivation and re-supply. Transcript data were analysed with MapMan software to identify coordinated, system-wide changes in metabolism and other cellular processes. Phosphorus (P) deprivation led to induction or repression of > 1000 genes involved in many processes. A subset, including the induction of genes involved in P uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of P-containing phospholipids with galactolipids and the repression of genes involved in nucleotide/nucleic acid synthesis, was reversed within 3 h after Pi re-supply. Analyses of 22 enzyme activities revealed that changes in transcript levels often, but not always, led to changes in the activities of the encoded enzymes in P-deprived plants. Analyses of metabolites confirmed that P deprivation leads to a shift towards the accumulation of carbohydrates, organic acids and amino acids, and that Pi re-supply leads to use of the latter. P-deprived plants also showed large changes in the expression of many genes involved in, for example, secondary metabolism and photosynthesis. These changes were not reversed rapidly upon Pi re-supply and were probably secondary in origin. Differentially expressed and highly P-specific putative regulator genes were identified that presumably play central roles in coordinating the complex responses of plants to changes in P nutrition. The specific responses to Pi differ markedly from those found for nitrate, whereas the long-term responses during P and N deprivation share common and non-specific features.

Arabidopsis↗

PubMed2006

PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments.

BACKGROUND: Microarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis. RESULTS: Here we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs.PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis.PageMan offers a complete user's guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/. CONCLUSION: PageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.

Database Management Systems↗

PubMed2006

Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior.

Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits. Finally, there was a strong relationship between ripening-associated transcripts and specific metabolite groups, such as TCA-cycle organic acids and sugar phosphates, underlining the importance of the respective metabolic pathways during fruit development.

Carbon↗

PubMed2006

Conversion of MapMan to allow the analysis of transcript data from Solanaceous species: effects of genetic and environmental alterations in energy metabolism in the leaf.

The tomato microarray TOM1 offers the possibility to monitor the levels of several thousand transcripts in parallel. The microelements represented on this tomato microarray have been putatively assigned to unigenes, and organised in functional classes using the MapMan ontology (Thimm et al., 2004. Plant J. 37: 914-939). This ontology was initially developed for use with the Arabidopsis ATH1 array, has a low level of redundancy, and can be combined with the MapMan software to provide a biologically structured overview of changes of transcripts, metabolites and enzyme activities. Use of this application is illustrated using three case studies with published or novel TOM1 array data sets for Solanaceous species. Comparison of previously reported data on transcript levels in potato leaves in the middle of the day and the middle of the night identified coordinated changes in the levels of transcripts of genes involved in various metabolic pathways and cellular events. Comparison with diurnal changes of gene expression in Arabidopsis revealed common features, illustrating how MapMan can be used to compare responses in different organisms. Comparison of transcript levels in new experiments performed on the leaves of the cultivated tomato S. lycopersicum and the wild relative S. pennellii revealed a general decrease of levels of transcripts of genes involved in terpene and, phenylpropanoid metabolism as well as chorismate biosynthesis in the crop compared to the wild relative. This matches the recently reported decrease of the levels of secondary metabolites in the latter. In the third case study, new expression array data for two genotypes deficient in TCA cycle enzymes is analysed to show that these genotypes have elevated levels of transcripts associated with photosynthesis. This in part explains the previously documented enhanced rates of photosynthesis in these genotypes. Since the Solanaceous MapMan is intended to be a community resource it will be regularly updated on improvements in tomato gene annotation and transcript profiling resources.

Arabidopsis↗

PubMed2005

Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis.

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO(2)]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.

Arabidopsis↗

PubMed2005

The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides.

The nucleotide sugar UDP-glucuronic acid (UDP-GlcA) is the principal precursor for galacturonic acid, xylose, apiose and arabinose residues of the plant cell-wall polymers. UDP-GlcA can be synthesized by two different functional pathways in Arabidopsis involving either UDP-glucose dehydrogenase or inositol oxygenase as the initial enzyme reaction to channel carbohydrates into a pool of UDP sugars used for cell-wall biosynthesis. The genes for the enzyme myo-inositol oxygenase (MIOX) were analyzed in Arabidopsis. They represent a small gene family containing four members. The transcription of all those members indicates a transient and organ-specific gene expression pattern in growing plant tissues as analyzed by RT-PCR and in promoter::GUS reporter gene lines. Two isoforms (MIOX1, MIOX2) are expressed in almost all tissues of the plant, whereas the expression of MIOX4 and MIOX5 is largely restricted to flowers, particularly maturing pollen. T-DNA insertion lines in MIOX genes were isolated; however, single knock-outs show growth phenotypes similar to the wild type. The monosaccharide composition of the cell wall in these mutants is not significantly changed compared to wild type plants. However, the incorporation of 3H-inositol into wall polymers of seedlings is greatly impaired in the mutant lines (Delta)MIOX1 and (Delta)MIOX2, which are the only isoforms that are expressed in seedlings.

Arabidopsis↗

PubMed2005

Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses.

MapMan is a user-driven tool that displays large genomics datasets onto diagrams of metabolic pathways or other processes. Here, we present new developments, including improvements of the gene assignments and the user interface, a strategy to visualize multilayered datasets, the incorporation of statistics packages, and extensions of the software to incorporate more biological information including visualization of corresponding genes and horizontal searches for similar global responses across large numbers of arrays.

Genes, Plant↗

PubMed2004

GMD@CSB.DB: the Golm Metabolome Database.

UNLABELLED: Metabolomics, in particular gas chromatography-mass spectrometry (GC-MS) based metabolite profiling of biological extracts, is rapidly becoming one of the cornerstones of functional genomics and systems biology. Metabolite profiling has profound applications in discovering the mode of action of drugs or herbicides, and in unravelling the effect of altered gene expression on metabolism and organism performance in biotechnological applications. As such the technology needs to be available to many laboratories. For this, an open exchange of information is required, like that already achieved for transcript and protein data. One of the key-steps in metabolite profiling is the unambiguous identification of metabolites in highly complex metabolite preparations from biological samples. Collections of mass spectra, which comprise frequently observed metabolites of either known or unknown exact chemical structure, represent the most effective means to pool the identification efforts currently performed in many laboratories around the world. Here we present GMD, The Golm Metabolome Database, an open access metabolome database, which should enable these processes. GMD provides public access to custom mass spectral libraries, metabolite profiling experiments as well as additional information and tools, e.g. with regard to methods, spectral information or compounds. The main goal will be the representation of an exchange platform for experimental research activities and bioinformatics to develop and improve metabolomics by multidisciplinary cooperation. AVAILABILITY: http://csbdb.mpimp-golm.mpg.de/gmd.html CONTACT: Steinhauser@mpimp-golm.mpg.de SUPPLEMENTARY INFORMATION: http://csbdb.mpimp-golm.mpg.de/

Database Management Systems↗

PubMed2004

CSB.DB: a comprehensive systems-biology database.

SUMMARY: The open access comprehensive systems-biology database (CSB.DB) presents the results of bio-statistical analyses on gene expression data in association with additional biochemical and physiological knowledge. The main aim of this database platform is to provide tools that support insight into life's complexity pyramid with a special focus on the integration of data from transcript and metabolite profiling experiments. The central part of CSB.DB, which we describe in this applications note, is a set of co-response databases that currently focus on the three key model organisms, Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana. CSB.DB gives easy access to the results of large-scale co-response analyses, which are currently based exclusively on the publicly available compendia of transcript profiles. By scanning for the best co-responses among changing transcript levels, CSB.DB allows to infer hypotheses on the functional interaction of genes. These hypotheses are novel and not accessible through analysis of sequence homology. The database enables the search for pairs of genes and larger units of genes, which are under common transcriptional control. In addition, statistical tools are offered to the user, which allow validation and comparison of those co-responses that were discovered by gene queries performed on the currently available set of pre-selectable datasets. AVAILABILITY: All co-response databases can be accessed through the CSB.DB Web server (http://csbdb.mpimp-golm.mpg.de/).

Database Management Systems↗

PubMed2004

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis.

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.

Arabidopsis↗

PubMed2003

RHM2 is involved in mucilage pectin synthesis and is required for the development of the seed coat in Arabidopsis.

Pectins are major components of primary plant cell walls and the seed mucilage of Arabidopsis. Despite progress in the structural elucidation of pectins, only very few enzymes participating in or regulating their synthesis have been identified. A first candidate gene involved in the synthesis of pectinaceous rhamnogalacturonan I is RHM2, a putative plant ortholog to NDP-rhamnose biosynthetic enzymes in bacteria. Expression studies with a promoter beta-glucuronidase construct and reverse transcription PCR data show that RHM2 is expressed ubiquitously. Rhm2 T-DNA insertion mutant lines were identified using a reverse genetics approach. Analysis of the rhm2 seeds by various staining methods and chemical analysis of the mucilage revealed a strong reduction of rhamnogalacturonan I in the mucilage and a decrease of its molecular weight. In addition, scanning electron microscopy of the seed surface indicated a distorted testa morphology, illustrating not only a structural but also a developmental role for RGI or rhamnose metabolism in proper testa formation.

Adhesives↗

PubMed2002

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.

Arabidopsis↗