The target of rapamycin (TOR) pathway is a major regulator of growth in all eukaryotes, integrating energy, nutrient and stress signals into molecular decisions. By using large-scale MS-based metabolite profiling of primary, secondary and lipid compounds in combination with array-based transcript profiling, we show that the TOR protein not only regulates growth but also influences nutrient partitioning and central energy metabolism. The study was performed on plants exhibiting conditional down-regulation of AtTOR expression, revealing strong regulation of genes involved in pathways such as the cell cycle, cell-wall modifications and senescence, together with major changes in transcripts and metabolites of the primary and secondary metabolism. In agreement with these results, our morphological and metabolic analyses disclosed major metabolic changes leading to massive accumulations of storage lipids and starch. The implications of these data in the context of the general role of TOR in eukaryotic systems are discussed in parallel with the plant-specific aspects of TOR function. Finally, we propose a role for harnessing the plant TOR pathway by utilizing it as a potent metabolic switch, offering a possible route for biotechnological optimization of plant energy content and carbon partitioning for the production of bioenergy.

SUMMARYThe time-resolved response of Arabidopsis thaliana towards changing light and/or temperature at the transcriptome and metabolome level is presented. Plants grown at 21°C with a light intensity of 150 lE m )2 sec )1 were either kept at this condition or transferred into seven different environments (4°C, darkness; 21°C, darkness; 32°C, darkness; 4°C, 85 lE m )2 sec). Samples were taken before (0 min) and at 22 time points after transfer resulting in (8·) 22 time points covering both a linear and a logarithmic time series totaling 177 states. Hierarchical cluster analysis shows that individual conditions (defined by temperature and light) diverge into distinct trajectories at condition-dependent times and that the metabolome follows different kinetics from the transcriptome. The metabolic responses are initially relatively faster when compared with the transcriptional responses. Gene Ontology over-representation analysis identifies a common response for all changed conditions at the transcriptome level during the early response phase (5-60 min). Metabolic networks reconstructed via metabolite-metabolite correlations reveal extensive environment-specific rewiring. Detailed analysis identifies conditional connections between amino acids and intermediates of the tricarboxylic acid cycle. Parallel analysis of transcriptional changes strongly support a model where in the absence of photosynthesis at normal/high temperatures protein degradation occurs rapidly and subsequent amino acid catabolism serves as the main cellular energy supply. These results thus demonstrate the engagement of the electron transfer flavoprotein system under short-term environmental perturbations.

In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

Starch represents the most important carbohydrate used for food and feed purposes. With the aim of increasing starch content, we decided to modulate the adenylate pool by changing the activity of the plastidial adenylate kinase in transgenic potato plants. As a result, we observed a substantial increase in the level of adenylates and, most importantly, an increase in the level of starch to 60% above that found in wild-type plants. In addition, concentrations of several amino acids were increased by a factor of 2-4. These results are particularly striking because this genetic manipulation also results in an increased tuber yield. The modulation of the plastidial adenylate kinase activity in transgenic plants therefore represents a potentially very useful strategy for increasing formation of major storage compounds in heterotrophic tissues of higher plants.