Yves Gibon

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 [CO2]. 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.

Extension of the visualization tool mapman to allow statistical analysis of arrays, display of coresponding 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 coresponding genes and horizontal searches for similar global responses across large numbers of arrays.

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.

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

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.

Starch as a major integrator in the regulation of plant growth

Rising demand for food and bioenergy makes it imperative to breed for increased crop yield. Vegetative plant growth could be driven by resource acquisition or developmental programs. Metabolite profiling in 94 Arabidopsis accessions revealed that biomass correlates negatively with many metabolites, especially starch. Starch accumulates in the light and is degraded at night to provide a sustained supply of carbon for growth. Multivariate analysis revealed that starch is an integrator of the overall metabolic response. We hypothesized that this reflects variation in a regulatory network that balances growth with the carbon supply. Transcript profiling in 21 accessions revealed coordinated changes of transcripts of more than 70 carbon-regulated genes and identified 2 genes (myo-inositol-1-phosphate synthase, a Kelch-domain protein) whose transcripts correlate with biomass. The impact of allelic variation at these 2 loci was shown by association mapping, identifying them as candidate lead genes with the potential to increase biomass production.

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.

Water deficits uncouple growth from photosynthesis, increase C content, and modify the relationships between C and growth in sink organs

In plants, carbon (C) molecules provide building blocks for biomass production, fuel for energy, and exert signalling roles to shape development and metabolism. Accordingly, plant growth is well correlated with light interception and energy conversion through photosynthesis. Because water deficits close stomata and thus reduce C entry, it has been hypothesised that droughted plants are under C starvation and their growth under C limitation. In this review, these points are questioned by combining literature review with experimental and modelling illustrations in various plant organs and species. First, converging evidence is gathered from the literature that water deficit generally increases C concentration in plant organs. The hypothesis is raised that this could be due to organ expansion (as a major C sink) being affected earlier and more intensively than photosynthesis (C source) and metabolism. How such an increase is likely to interact with C signalling is not known. Hence, the literature is reviewed for possible links between C and stress signalling that could take part in this interaction. Finally, the possible impact of water deficit-induced C accumulation on growth is questioned for various sink organs of several species by combining published as well as new experimental data or data generated using a modelling approach. To this aim, robust correlations between C availability and sink organ growth are reported in the absence of water deficit. Under water deficit, relationships weaken or are modified suggesting release of the influence of C availability on sink organ growth. These results are interpreted as the signature of a transition from source to sink growth limitation under water deficit.

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

BackgroundMicroarray 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.ResultsHere 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 users guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/.ConclusionPageMan 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.

Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in Arabidopsis rosettes

BackgroundGenome-wide transcript profiling and analyses of enzyme activities from central carbon and nitrogen metabolism show that transcript levels undergo marked and rapid changes during diurnal cycles and after transfer to darkness, whereas changes in activities are smaller and delayed. In the starchless pgm mutant, where sugars are depleted every night, there are accentuated diurnal changes in transcript levels. Enzyme activities in this mutant do not show larger diurnal changes; instead, they shift towards the levels found in the wild type after several days of darkness. This indicates that enzyme activities change slowly, integrating the changes in transcript levels over several diurnal cycles.ResultsTo generalize this conclusion, 137 metabolites were profiled using gas and liquid chromatography coupled to mass spectroscopy. The amplitudes of the diurnal changes in metabolite levels in pgm were (with the exception of sugars) similar or smaller than in the wild type. The average levels shifted towards those found after several days of darkness in the wild type. Examples include increased levels of amino acids due to protein degradation, decreased levels of fatty acids, increased tocopherol and decreased myo-inositol. Many metabolite-transcript correlations were found and the proportion of transcripts correlated with sugars increased dramatically in the starchless mutant.ConclusionRapid diurnal changes in transcript levels are integrated over time to generate quasi-stable changes across large sectors of metabolism. This implies that correlations between metabolites and transcripts are due to regulation of gene expression by metabolites, rather than metabolites being changed as a consequence of a change in gene expression.