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Mark Stitt

Publications and source records attributed to Mark Stitt.

36 records · Page 2Linked to original sources
PubMed2005

Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis.

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.

Arabidopsis↗
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

A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness.

A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to show that, irrespective of the time of harvest during the diurnal cycle, the pgm mutant resembles a wild-type plant that has been exposed to a 3 d prolongation of the night.

Arabidopsis↗
PubMed2004

The role of raffinose in the cold acclimation response of Arabidopsis thaliana.

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.

Acclimatization↗
PubMed2004

Metabolite profiling in plant biology: platforms and destinations.

Optimal use of genome sequences and gene-expression resources requires powerful phenotyping platforms, including those for systematic analysis of metabolite composition. The most used technologies for metabolite profiling, including mass spectral, nuclear magnetic resonance and enzyme-based approaches, have various advantages and disadvantages, and problems can arise with reliability and the interpretation of the huge datasets produced. These techniques will be useful for answering important biological questions in the future.

Biology↗
PubMed2004

Target-based discovery of novel herbicides.

In the past 10 years, strategies for the first steps of herbicide discovery have switched from the testing of chemicals for efficacy on whole plants towards the use of in-vitro assays against molecular targets. Many different approaches have been developed to identify bona fide targets for in-vitro screening. Developments in functional genomics and in pharmaceutical research could aid the development of assay systems for the evaluation of chemicals for their suitability as lead structures in herbicide discovery.

Agrochemicals↗
PubMed2004

Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen.

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

Arabidopsis↗
PubMed2004

MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes.

MAPMAN is a user-driven tool that displays large data sets onto diagrams of metabolic pathways or other processes. SCAVENGER modules assign the measured parameters to hierarchical categories (formed 'BINs', 'subBINs'). A first build of TRANSCRIPTSCAVENGER groups genes on the Arabidopsis Affymetrix 22K array into >200 hierarchical categories, providing a breakdown of central metabolism (for several pathways, down to the single enzyme level), and an overview of secondary metabolism and cellular processes. METABOLITESCAVENGER groups hundreds of metabolites into pathways or groups of structurally related compounds. An IMAGEANNOTATOR module uses these groupings to organise and display experimental data sets onto diagrams of the users' choice. A modular structure allows users to edit existing categories, add new categories and develop SCAVENGER modules for other sorts of data. MAPMAN is used to analyse two sets of 22K Affymetrix arrays that investigate the response of Arabidopsis rosettes to low sugar: one investigates the response to a 6-h extension of the night, and the other compares wild-type Columbia-0 (Col-0) and the starchless pgm mutant (plastid phosphoglucomutase) at the end of the night. There were qualitatively similar responses in both treatments. Many genes involved in photosynthesis, nutrient acquisition, amino acid, nucleotide, lipid and cell wall synthesis, cell wall modification, and RNA and protein synthesis were repressed. Many genes assigned to amino acid, nucleotide, lipid and cell wall breakdown were induced. Changed expression of genes for trehalose metabolism point to a role for trehalose-6-phosphate (Tre6P) as a starvation signal. Widespread changes in the expression of genes encoding receptor kinases, transcription factors, components of signalling pathways, proteins involved in post-translational modification and turnover, and proteins involved in the synthesis and sensing of cytokinins, abscisic acid (ABA) and ethylene revealing large-scale rewiring of the regulatory network is an early response to sugar depletion.

Abscisic Acid↗
PubMed2004

Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes.

Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.

Arabidopsis↗
PubMed2004

Adjustment of diurnal starch turnover to short days: depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period.

A larger proportion of the fixed carbon is retained as starch in the leaf in short days, providing a larger store to support metabolism and carbon export during the long night. The mechanisms that facilitate this adjustment of the sink-source balance are unknown. Starchless pgm mutants were analysed to discover responses that are triggered when diurnal starch turnover is disturbed. Sugars accumulated to high levels during the day, and fell to very low levels by the middle of the night. Sugars rose rapidly in the roots and rosette after illumination, and decreased later in the light period. Global transcript profiling revealed only small differences between pgm and Col0 at the end of the day but large differences at the end of the night, when pgm resembled Col0 after a 4-6 h prolongation of the night and many genes required for biosynthesis and growth were repressed [Plant J. 37 (2004) 914]. It is concluded that transient sugar depletion at the end of the night inhibits carbon utilization at the start of the ensuing light period. A second set of experiments investigated the stimulation of starch synthesis in response to short days in wild-type Col0. In short days, sugars were very low in the roots and rosette at the end of the dark period, and after illumination accumulated rapidly in both organs to levels that were higher than in long days. The response resembles pgm, except that carbohydrate accumulated in the leaf as starch instead of sugars. A similar response was found after transfer from long to short days. Inclusion of sugar in the rooting medium attenuated the stimulation of starch synthesis. Post-translational activation of ADP-glucose pyrophosphorylase (AGPase) was increased in pgm, and in Col0 in short days. It is concluded that starch synthesis is stimulated in short day conditions because sugar depletion at the end of the night triggers a temporary inhibition of growth and carbohydrate utilization in the first part of the light period, leading to transient accumulation of sugar and activation of AGPase.

Arabidopsis↗
PubMed2003

ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species.

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed reaction in the pathway of starch synthesis. It was recently shown that potato (Solanum tuberosum) tuber AGPase is subject to redox-dependent posttranslational regulation, involving formation of an intermolecular Cys bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme (A. Tiessen, J.H.M. Hendriks, M. Stitt, A. Branscheid, Y. Gibon, E.M. Farré, P. Geigenberger [2002] Plant Cell 14: 2191-2213). We show here that AGPase is also subject to posttranslational regulation in leaves of pea (Pisum sativum), potato, and Arabidopsis. Conversion is accompanied by an increase in activity, which involves changes in the kinetic properties. Light and sugars act as inputs to trigger posttranslational regulation of AGPase in leaves. AGPB is rapidly converted from a dimer to a monomer when isolated chloroplasts are illuminated and from a monomer to a dimer when preilluminated leaves are darkened. AGPB is converted from a dimer to monomer when sucrose is supplied to leaves via the petiole in the dark. Conversion to monomeric form increases during the day as leaf sugars increase. This is enhanced in the starchless phosphoglucomutase mutant, which has higher sugar levels than wild-type Columbia-0. The extent of AGPB monomerization correlates with leaf sugar levels, and at a given sugar content, is higher in the light than the dark. This novel posttranslational regulation mechanism will allow starch synthesis to be regulated in response to light and sugar levels in the leaf. It complements the well-characterized regulation network that coordinates fluxes of metabolites with the recycling of phosphate during photosynthetic carbon fixation and sucrose synthesis.

Amino Acid Sequence↗
PubMed2003

From measurements of metabolites to metabolomics: an 'on the fly' perspective illustrated by recent studies of carbon-nitrogen interactions.

Techniques for surveying metabolite levels provide a powerful tool for basic plant research and biotechnology. They offer the possibility of specialised measurements of specific classes of metabolites, profiling of a very broad range of low molecular weight compounds, and resolving the spatial and/or temporal localisation of selected key metabolites. Recent applications in the field of carbon-nitrogen interactions provide a framework to discuss how metabolite datasets are being utilised in the post-genomic era to characterise system responses, link transcript data to phenotypic responses, analyse underlying regulation mechanisms, and implement an in-context analysis of gene function. A major future challenge concerns the integration of the information gained from metabolite profiling into an accessible body of knowledge.

Carbon↗
PubMed2002

A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis.

Low temperatures lead to the inhibition of sucrose synthesis and photosynthesis. The biochemical and physiological adaptations of plants to low temperatures include the post-translational activation and increased expression of enzymes of the sucrose synthesis pathway, the changed expression of Calvin cycle enzymes, and changes in the leaf protein content. Recent progress has been made in understanding both the signals that trigger these processes and how the regulation of photosynthetic carbon metabolism interacts with other processes during cold acclimation.

Acclimatization↗
PubMed2002

Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.

Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.

Adenine Nucleotides↗
PubMed2002

Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants.

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon- and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild-type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.

Amino Acids↗
PubMed2002

Steps towards an integrated view of nitrogen metabolism.

This article discusses how nitrate assimilation is integrated with nitrate uptake, with ammonium assimilation and amino acid synthesis, with pH regulation, and with the sugar supply in tobacco leaves. During the first part of the light period, nitrate assimilation exceeds nitrate uptake by 2-fold and ammonium assimilation by 50%, leading to rapid depletion of nitrate and accumulation of ammonium, glutamine, glycine and serine. NIA, NII and PPC expression show a shared maximum early in the diurnal cycle to direct carbon towards malate synthesis for pH regulation. Later in the diurnal cycle an orchestrated increase of GLN2, PKc, CS, and ICDH-1 expression re-establishes a balance between nitrate assimilation and ammonium metabolism. Nitrate uptake continues throughout the night, replenishing the leaf nitrate pool. These diurnal changes are attenuated or abolished in mutants with low NIA activity, and modified in wild-type plants growing on different nitrogen sources or elevated [CO(2)]. Comparison across genotypes and conditions reveals that NIA transcript levels are always closely related to the balance between nitrate influx and assimilation, but are unrelated to changes of glutamine or 2-oxoglutarate. In a systematic search for other downstream regulators, a wide range of downstream metabolites was fed to detached leaves and glutamate, cysteine, asparagine, and malate identified as candidates. Low sugars totally inhibit nitrate assimilation, overriding signals from nitrogen metabolism. Moderate changes act post-transcriptionally, and larger changes lead to a collapse of the NIA transcript. Low sugars also lead to a collapse of minor amino acids and a dramatic decrease of phenylpropanoids and nicotine. Consequently, wild-type plants growing in unfavourable light regimes and antisense RBCS transformants are simultaneously carbon- and nitrogen-limited.

Amino Acid Oxidoreductases↗
PubMed2002

Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply.

Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.

Chloroplasts↗