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.

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.

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.

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

BACKGROUND: Genome-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. RESULTS: To 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. CONCLUSION: Rapid 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.

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.

Metabolite profiling as an aid to metabolic engineering in plants.

The past decade has seen some impressive successes in the metabolic engineering of biotechnologically important plant pathways. However, plant metabolic engineering currently proceeds more by trial and error than by intelligent system design. A change in philosophy away from studying pathways in isolation and towards studying metabolism as a network is necessary. To support this development, improvements in technologies for metabolic analysis, a wider adoption of metabolite-profiling approaches and significant innovations in data analysis methodologies are required.

Potato hexokinase 2 complements transgenic Arabidopsis plants deficient in hexokinase 1 but does not play a key role in tuber carbohydrate metabolism.

Potato plants (Solanum tuberosum L. cv. Désirée) transformed with sense and antisense constructs of a cDNA encoding the potato hexokinase 2 exhibited altered enzyme activities and expression of hexokinase 2 mRNA. Measurements of the maximum catalytic activity of hexokinase revealed an 11-fold variation in leaf (from 48% of the wild-type activity in antisense transformants to 446% activity in sense transformants) and an 8-fold variation in developing tubers (from 35% of the wild-type activity in antisense transformants to 212% activity in sense transformants). Despite the wide range of hexokinase activities, no substantial change was found in the fresh weight yield, starch, sugar and metabolite levels of transgenic tubers. However, both potato hexokinases 1 and 2 were able to complement the hyposensitivity of antisense hexokinase 1 Arabidopsis transgenic plants to glucose. In an in vitro bioassay of seed germination in a medium with high glucose levels, double transformants showed the same sensitivity to glucose as that of the wild-type ecotype, displaying a stunted phenotype in hypocotyls, cotyledons and roots.

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism.

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.

Sucrose to starch: a transition in molecular plant physiology.

The major flux in potato tuber carbon metabolism is the conversion of sucrose through hexose phosphates to starch. The enzymes that mediate this pathway are well characterized, the genes that encode them have been cloned and transgenic plants have been generated. These transgenic studies have confirmed hypotheses based on more indirect methods, but they have also generated new challenges by highlighting the enormous flexibility and complexity inherent in plant metabolism. The investigation of the sucrose-to-starch transition in potato tubers is an excellent example of how the discipline of molecular plant physiology is evolving at both the scientific and technical levels.

The control of source to sink carbon flux during tuber development in potato.

We have used top-down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of 14 C from CO2 to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.