Reproductive failure in Arabidopsis thaliana under transient carbohydrate limitation: flowers and very young siliques are jettisoned and the meristem is maintained to allow successful resumption of reproductive growth.

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again.

Xeml Lab: a tool that supports the design of experiments at a graphical interface and generates computer-readable metadata files, which capture information about genotypes, growth conditions, environmental perturbations and sampling strategy.

Data mining depends on the ability to access machine-readable metadata that describe genotypes, environmental conditions, and sampling times and strategy. This article presents Xeml Lab. The Xeml Interactive Designer provides an interactive graphical interface at which complex experiments can be designed, and concomitantly generates machine-readable metadata files. It uses a new eXtensible Mark-up Language (XML)-derived dialect termed XEML. Xeml Lab includes a new ontology for environmental conditions, called Xeml Environment Ontology. However, to provide versatility, it is designed to be generic and also accepts other commonly used ontology formats, including OBO and OWL. A review summarizing important environmental conditions that need to be controlled, monitored and captured as metadata is posted in a Wiki (http://www.codeplex.com/XeO) to promote community discussion. The usefulness of Xeml Lab is illustrated by two meta-analyses of a large set of experiments that were performed with Arabidopsis thaliana during 5 years. The first reveals sources of noise that affect measurements of metabolite levels and enzyme activities. The second shows that Arabidopsis maintains remarkably stable levels of sugars and amino acids across a wide range of photoperiod treatments, and that adjustment of starch turnover and the leaf protein content contribute to this metabolic homeostasis.

Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range.

This paper characterizes the transcriptional and metabolic response of a chilling-tolerant species to an increasingly large decrease of the temperature. Arabidopsis Col-0 was grown at 20 degrees C and transferred to 17, 14, 12, 10 or 8 degrees C for 6 and 78 h, before harvesting the rosette and profiling >22 000 transcripts, >20 enzyme activities and >80 metabolites. Most parameters showed a qualitatively similar response across the entire temperature range, with the amplitude increasing as the temperature decreased. Transcripts typically showed large changes after 6 h, which were often damped by 78 h. Genes were induced for sucrose, proline, raffinose, tocopherol and polyamine synthesis, phenylpropanoid and flavonoid metabolism, fermentation, non-phosphorylating mitochondrial electron transport, RNA processing, and protein synthesis, targeting and folding. Genes were repressed for carbonic anhydrases, vacuolar invertase, and ethylene and jasmonic acid signalling. While some enzyme activities and metabolites changed rapidly, most changed slowly. After 6 h, there was an accumulation of phosphorylated intermediates, a shift of partitioning towards sucrose, and a perturbation of glycine decarboxylation and nitrogen metabolism. By 78 h, there was an increase of the overall protein content and many enzyme activities, a general increase of carbohydrates, organic and amino acids, and an increase of many stress-responsive metabolites including raffinose, proline, tocopherol and polyamines. When the responses of transcripts and metabolism were compared, there was little agreement after 6 h, but considerable agreement after 78 h. Comparison with the published studies indicated that much, but not all, of the response was orchestrated by the CBF programme. Overall, our results showed that transcription and metabolism responded in a continuous manner across a wide range of temperatures. The general increase of enzyme activities and metabolites emphasized the positive and compensatory nature of this response.

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.

Serine 774 and amino acids 296 to 437 comprise the major C4 determinants of the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia.

C4 phosphoenolpyruvate carboxylases have evolved several times independently from non-photosynthetic C3 ancestral enzymes. To identify C4-specific determinants at the amino acid level the two orthologous ppcA PEPCases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were used as a model system. In a previous publication [Bläsing et al., J. Biol. Chem. 275 (2000) 27917-27923] it was reported that the serine at position 774 is an invariant residue in all C4 phosphoenolpyruvate carboxylases. Here we show by swapping experiments and site-directed mutagenesis that the serine 774 and amino acids 296-437 explain two thirds of the C4 characteristic phosphoenolpyruvate saturation kinetics when investigated in the C3 background. In addition, the results indicate that the determinants functionally interact with each other.

A fluorometric assay for trehalose in the picomole range.

BACKGROUND: Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity. RESULTS: A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively. CONCLUSION: The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Global transcript levels respond to small changes of the carbon status during progressive exhaustion of carbohydrates in Arabidopsis rosettes.

The balance between the supply and utilization of carbon (C) changes continually. It has been proposed that plants respond in an acclimatory manner, modifying C utilization to minimize harmful periods of C depletion. This hypothesis predicts that signaling events are initiated by small changes in C status. We analyzed the global transcriptional response to a gradual depletion of C during the night and an extension of the night, where C becomes severely limiting from 4 h onward. The response was interpreted using published datasets for sugar, light, and circadian responses. Hundreds of C-responsive genes respond during the night and others very early in the extended night. Pathway analysis reveals that biosynthesis and cellular growth genes are repressed during the night and genes involved in catabolism are induced during the first hours of the extended night. The C response is amplified by an antagonistic interaction with the clock. Light signaling is attenuated during the 24-h light/dark cycle. A model was developed that uses the response of 22K genes during a circadian cycle and their responses to C and light to predict global transcriptional responses during diurnal cycles of wild-type and starchless pgm mutant plants and an extended night in wild-type plants. By identifying sets of genes that respond at different speeds and times during C depletion, our extended dataset and model aid the analysis of candidates for C signaling. This is illustrated for AKIN10 and four bZIP transcription factors, and sets of genes involved in trehalose signaling, protein turnover, and starch breakdown.

Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings.

Arabidopsis seedlings were subjected to 2 days of carbon starvation, and then resupplied with 15 mm sucrose. The transcriptional and metabolic response was analyzed using ATH1 arrays, real-time quantitative (q)RT-PCR analysis of >2000 transcription regulators, robotized assays of enzymes from central metabolism and metabolite profiling. Sucrose led within 30 min to greater than threefold changes of the transcript levels for >100 genes, including 20 transcription regulators, 15 ubiquitin-targeting proteins, four trehalose phosphate synthases, autophagy protein 8e, several glutaredoxins and many genes of unknown function. Most of these genes respond to changes of endogenous sugars in Arabidopsis rosettes, making them excellent candidates for upstream components of sugar signaling pathways. Some respond during diurnal cycles, consistent with them acting in signaling pathways that balance the supply and utilization of carbon in normal growth conditions. By 3 h, transcript levels change for >1700 genes. This includes a coordinated induction of genes involved in carbohydrate synthesis, glycolysis, respiration, amino acid and nucleotide synthesis, DNA, RNA and protein synthesis and protein folding, and repression of genes involved in amino acid and lipid catabolism, photosynthesis and chloroplast protein synthesis and folding. The changes of transcripts are followed by a delayed activation of central metabolic pathways and growth processes, which use intermediates from these pathways. Sucrose and reducing sugars accumulate during the first 3-8 h, and starch for 24 h, showing that there is a delay until carbon utilization for growth recommences. Gradual changes of enzyme activities and metabolites are found for many metabolic pathways, including glycolysis, nitrate assimilation, the shikimate pathway and myoinositol, proline and fatty acid metabolism. After 3-8 h, there is a decrease of amino acids, followed by a gradual increase of protein.

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.

Evolution of C4 phosphoenolpyruvate carboxylase.

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phosphoenolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme's kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.

Molecular evolution of C4 phosphoenolpyruvate carboxylase in the genus Flaveria--a gradual increase from C3 to C4 characteristics.

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.

The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia -- implications for the evolution of C4 photosynthesis.

C4 phospho enolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K(0.5)-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K(0.5)-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k(cat). We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.

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.