Biomedical subjects

Lothar Willmitzer

Publications and source records attributed to Lothar Willmitzer.

At least 19 recordsLinked to original sources

PubMed2007

Integrating profiling data: using linear correlation to reveal coregulation of transcript and metabolites.

Recent advances in the medical and biological sciences have been characterized by a major paradigm shift from reductionism to integrated and holistic systems approaches. Such approaches are characterized at the experimental level by the multiparallel analysis of a multitude of parameters of a given biological system at a range of different molecular levels, following the systematic perturbation of the system in question. Although a multitude of studies have been carried out to assess the transcript, protein, and metabolite complements of cells under various conditions, to date, few have been attempted that encompass the profiling of more than one of these entities. In this chapter, we describe combined analysis of data obtained from transcript and metabolic profiling, and detail advantages of using both approaches in parallel.

Gas Chromatography-Mass Spectrometry↗

PubMed2006

Subcellular pyrophosphate metabolism in developing tubers of potato (Solanum tuberosum).

PPi has previously been implicated specifically in the co-ordination of the sucrose-starch transition and in the broader context of its role as co-factor in heterotrophic plant metabolism. In order to assess the compartmentation of pyrophosphate (PPi) metabolism in the potato tuber we analysed the effect of expressing a bacterial pyrophosphatase in the amyloplast of wild type tubers or in the cytosol or amyloplast of invertase-expressing tubers. The second and third approaches were adopted since we have previously characterized the invertase expressing lines to both exhibit highly altered sucrose metabolism and to contain elevated levels of PPi (Farré et al. (2000a) Plant Physiol 123:681) and therefore this background rendered questions concerning the level of communication between the plastidic and cytosolic pyrophosphate pools relatively facile. In this study we observed that the increase in PPi in the invertase expressing lines was mainly confined to the cytosol. Accordingly, the expression of a bacterial pyrophosphatase in the plastid of either wild type or invertase-expressing tubers did not lead to a decrease in total PPi content. However, the expression of the heterologous pyrophosphatase in the cytosol of cytosolic invertase-expressing tubers led to strong metabolic changes. These results are discussed both with respect to our previous hypotheses and to current models of the compartmentation of potato tuber metabolism.

Bacterial Proteins↗

PubMed2006

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement.

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.

Computer Simulation↗

PubMed2005

Enhancing vacuolar sucrose cleavage within the developing potato tuber has only minor effects on metabolism.

Modification of tuber carbohydrate metabolism by the tuber-specific expression of a yeast invertase targeted to the cytosol or apoplast has previously been demonstrated to have diverse effects on tuber growth and metabolism. In the current study, we generated plants exhibiting tuber-specific expression of the same enzyme targeted to the vacuole. Enzymatic analysis of the carbohydrate levels of the tuber revealed dramatic decreases in sucrose content coupled with large increases in the levels of glucose and hexose phosphates, but unaltered starch content in the transformants. Analysis of the key enzyme of glycolysis suggests that this pathway is down-regulated in the transformants. Despite these changes in metabolite pools and enzyme activity, few consistent changes could be observed in the estimated metabolic fluxes following incubation of isolated tuber discs in labelled glucose. The analysis of the relative levels of a wide range of metabolites using a gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling method revealed large changes in the levels of fructose and decreases in a range of other sugars, but very few changes in the contents of organic and amino acids. This metabolic profile is remarkably consistent with that obtained following expression of the invertase in the apoplastic compartment, providing circumstantial evidence for the endocytotic trafficking of sugars within potato tuber parenchyma. Finally, the results of this study are compared with those from other plant species and the relative roles of the vacuolar isoform of the enzyme are contrasted.

Carbohydrate Metabolism↗

PubMed2005

Integrative gene-metabolite network with implemented causality deciphers informational fluxes of sulphur stress response.

The systematic accumulation of gene expression data, although revolutionary, is insufficient in itself for an understanding of system-level physiology. In the post-genomic era, the next cognitive step is linking genes to biological processes and assembling a mosaic of data into global models of biosystem function. A dynamic network of informational flows in Arabidopsis plants perturbed by sulphur depletion is presented here. With the use of an original protocol, the first biosystem response network was reconstructed from a time series of transcript and metabolite profiles, which, on the one hand, integrates complex metabolic and transcript data and, on the other hand, possesses a causal relationship. Using the informational fluxes within this reconstruction, it was possible to link system perturbation to response endpoints. Robustness and stress tolerance, as consequences of scale-free network topology, and hubs, as potential controllers of homeostasis maintenance, were revealed. Communication paths of propagating system excitement directed to physiological endpoints, such as anthocyanin accumulation and enforced root formation were dissected from the network. An auxin regulatory circuit involved in the control of a hypo-sulphur stress response was uncovered.

Arabidopsis↗

PubMed2005

Modulation of fructokinase activity of potato (Solanum tuberosum) results in substantial shifts in tuber metabolism.

Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

Carbohydrate Metabolism↗

PubMed2005

Expression of an Escherichia coli phosphoglucomutase in potato (Solanum tuberosum L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting.

The aim of this work was to evaluate the influence of elevating the cytosolic activity of phosphoglucomutase (PGM; EC 5.4.2.2) on photosynthesis, growth and heterotrophic metabolism. Here we describe the generation of novel transgenic plants expressing an Escherichia coli phosphoglucomutase (EcPGM) under the control of the 35S promoter. These lines were characterised by an accumulation of leaf sucrose, despite displaying no alterations in photosynthetic carbon partitioning, and a reduced tuber starch content. Determinations of the levels of a wide range of other metabolites revealed dramatic reductions in maltose and other sugars in leaves of the transformants, as well as a modification of the pattern of organic and amino acid content in tubers of these lines. Intriguingly, the transgenics also displayed a dramatically delayed rate of sprouting and significantly enhanced rate of respiration, however, it is important to note that the severity of these traits did not always correlate with the level of transgene expression. These results are discussed in the context of current understanding of the control of respiration and the breaking of tuber dormancy.

Carbohydrate Metabolism↗

PubMed2005

Tuber-specific cytosolic expression of a bacterial phosphoglucomutase in potato (Solanum tuberosum L.) dramatically alters carbon partitioning.

Constitutive antisense inhibition of the cytosolic isoform of phosphoglucomutase in the potato (Solanum tuberosum L.) results in restriction of photosynthesis, growth inhibition and modified tuber morphology, and a severe restriction of tuber starch synthesis. Here we describe the consequences of the tuber-specific expression of an Escherichia coli phosphoglucomutase in the cytosol. Analysis of [14C]glucose metabolism by tuber discs isolated from wild type and transformants revealed that the rates of sucrose and starch synthesis were unaltered but that the rate of glycolysis was depressed in the transgenics. The transformant tubers also contained dramatically reduced amino acid content and significantly higher levels of ADP, but were characterized by elevated levels of Krebs cycle intermediates and an unaltered rate of respiration. In addition to these metabolic consequences of the overexpression of the E. coli enzyme, we observed morphological changes in tubers, with the transformants having a smaller number of larger tubers which exhibited delayed rates of sprouting with respect to the wild type. These results are discussed with respect to current models of the regulation of central plant metabolism and tuber dormancy.

Adenosine Monophosphate↗

PubMed2005

GC-MS libraries for the rapid identification of metabolites in complex biological samples.

Gas chromatography-mass spectrometry based metabolite profiling of biological samples is rapidly becoming one of the cornerstones of functional genomics and systems biology. Thus, the technology needs to be available to many laboratories and open exchange of information is required such as those achieved for transcript and protein data. The key-step in metabolite profiling is the unambiguous identification of metabolites in highly complex metabolite preparations with composite structure. Collections of mass spectra, which comprise frequently observed identified and non-identified metabolites, represent the most effective means to pool the identification efforts currently performed in many laboratories around the world. Here, we describe a platform for mass spectral and retention time index libraries that will enable this process (MSRI; www.csbdb.mpimp-golm.mpg.de/gmd.html). This resource should ameliorate many of the problems that each laboratory will face both for the initial establishment of metabolome analysis and for its maintenance at a constant sample throughput.

Amino Acids↗

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

Towards dissecting nutrient metabolism in plants: a systems biology case study on sulphur metabolism.

A genomics analysis on sulphur metabolism has been conducted at the level of transcriptomics and metabolomics. The analysis of these data after applying bioinformatic tools is to reveal novel findings. The findings are discussed and the knowledge obtained from comparable analyses on sulphur metabolism and other plant nutrient genomic studies is reviewed. The analysis of the response of the transcriptome and metabolome to sulphur deprivation in the growth medium provides a tool set for the analysis of comparable genomics studies of other nutrients. The goal of this 'sulphobolomics' (i.e. sulphur genomics and metabolome analysis) approach, and of other investigations, is to describe in a holistic way the biochemical, molecular, and physiological response of a plant to nutrient starvation, here sulphate, or, more generally, to alterations and imbalances in nutrient availability. Eventually, this analysis will provide a case study for a systems biology approach.

Arabidopsis↗

PubMed2004

Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Acetate-CoA Ligase↗

PubMed2004

Temporally regulated expression of a yeast invertase in potato tubers allows dissection of the complex metabolic phenotype obtained following its constitutive expression.

The constitutive cytosolic expression of a yeast ( Saccharomyces cerevisiae ) invertase within potato ( Solanum tuberosum ) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis. These transgenic lines were further characterised by a massive cycle of sucrose degradation and resynthesis via sucrose-phosphate synthase. We have recently developed a B33 patatin driven alc gene construct allowing tight chemical control of gene expression following supply of acetaldehyde with minimal pleiotropic effects of the inducing agent on metabolism. This construct was used for chemical induction of the yeast invertase gene after 10-weeks growth to dissect the complex metabolic phenotype obtained after constitute expression. Inducible expression led to increased invertase activity within 24 h in well-defined areas within growing tubers. Although the sucrose levels were reduced, there was no effect on the levels of starch whilst levels of many amino acids decreased. Labelling experiments revealed that these lines exhibited increased rates of sucrose cycling, whereas rates of glycolysis and of starch synthesis were not substantially changed. From these results we conclude that sucrose cycling is stimulated in response to a short-term increase in the rate of sucrose mobilisation, providing evidence for a role of sucrose cycling as a buffering capacity that regulates the net rate of sucrose usage. In contrast, the dramatic increase in hexose-phosphate levels and the switch from starch synthesis to respiration seen on the constitutive expression of the invertase was not observed in the inducible lines, suggesting that this is the result of cumulative pleiotropic effects that occurred when the transgene was expressed throughout development.

Carbon Radioisotopes↗

PubMed2004

Metabolite profiling: from diagnostics to systems biology.

The concept of metabolite profiling has been around for several decades, but only recent technical innovations have allowed metabolite profiling to be carried out on a large scale - with respect to both the number of metabolites measured and the number of experiments carried out. As a result, the power of metabolite profiling as a technology platform for diagnostics, and the research areas of gene-function analysis and systems biology, is now beginning to be fully realized.

Cell Physiological Phenomena↗

PubMed2004

Analysis of the transient increase in cytosolic Ca2+ during the action potential of higher plants with high temporal resolution: requirement of Ca2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression.

Plants respond to various abiotic stimuli by activation and propagation of fast electrical signals, action potentials. To resolve the temporal increase in cytosolic Ca(2)(+) during the action potentials of higher plants, we regenerated transgenic potato plants that expressed the Ca(2)(+) photoprotein apoaequorin. These genetically engineered potato plants were used for simultaneous measurements of transient changes in the membrane potential and the Ca(2)(+) luminescence triggered by heat-induced action potentials. High temporal resolution for recording of the fast transient electrical and light signals was accomplished by a sampling rate of 1 kHz. Upon elicitation by heat the membrane potential depolarization preceded the rise of cytosolic Ca(2)(+) by 50-100 ms. Several Ca(2)(+) channel blockers were tested to inhibit the rise in cytosolic Ca(2)(+). Treatment of plants with Ruthenium Red blocked the elevation in cytosolic Ca(2)(+) that was associated with heat-stimulated action potentials. Furthermore, action potentials have been demonstrated to stimulate jasmonic acid biosynthesis and PINII gene expression. Therefore, we measured jasmonic acid and PINII gene expression levels subsequent to action potential initiation by a short heating pulse. As expected, jasmonic acid biosynthesis and PINII gene expression were induced by action potentials. Pretreatment of potato plants with Ruthenium Red inhibited induction of jasmonic acid biosynthesis and PINII gene expression that was generally triggered by heat-activated action potentials.

Action Potentials↗

PubMed2004

Cell wall invertase expression at the apical meristem alters floral, architectural, and reproductive traits in Arabidopsis thaliana.

Resource allocation is a major determinant of plant fitness and is influenced by external as well as internal stimuli. We have investigated the effect of cell wall invertase activity on the transition from vegetative to reproductive growth, inflorescence architecture, and reproductive output, i.e. seed production, in the model plant Arabidopsis thaliana by expressing a cell wall invertase under a meristem-specific promoter. Increased cell wall invertase activity causes accelerated flowering and an increase in seed yield by nearly 30%. This increase is caused by an elevation of the number of siliques, which results from enhanced branching of the inflorescence. On the contrary, as cytosolic enzyme, the invertase causes delayed flowering, reduced seed yield, and branching. This demonstrates that invertases not only are important in determining sink strength of storage organs but also play a role in regulating developmental processes.

Arabidopsis↗

PubMed2004

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes.

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.

Amino Acids↗

PubMed2003

Expression of a yeast-derived invertase in companion cells results in long-distance transport of a trisaccharide in an apoplastic loader and influences sucrose transport.

Companion cell-specific expression of a cytosolic invertase from yeast ( Saccharomyces cerevisiae) was used as a tool to synthesise oligosaccharides in the sieve element/companion cell complex and study whether oligosaccharides could be transported in the phloem of an apoplastically loading species. Potato ( Solanum tuberosum L.) plants expressing the invertase under the control of the Agrobacterium tumefaciens rolC promoter produced the trisaccharide 6-kestose in leaves, which was transported via the phloem and accumulated in tubers of transgenic plants. In graft experiments with rolC invertase plants as scion and wild-type rootstocks, 6-kestose accumulated in tubers to levels comparable to sucrose. This shows that long-distance transport of oligosaccharides is possible in apoplastically loading plants, which normally transport only sucrose. The additional transport route for assimilates neither led to elevated photosynthetic activity nor to increased tuber yield. Enhanced sucrose turnover in companion cells caused large amounts of glucose and fructose to be exuded from leaf petioles, and elevated levels of sucrose were detected in phloem exudates. While the latter indicates a higher capacity for sucrose loading into the phloem due to increased metabolic activity of companion cells, the massive release of hexoses catalysed by the invertase seemed to interfere with assimilate delivery to sink organs.

Biological Transport↗