Nicolas Schauer

Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography–linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS–based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300–500 analytes of polar and nonpolar extracts in ∼400 experimental samples per week per machine.

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.

GC–MS libraries for the rapid identification of metabolites in complex biological samples

Gas chromatography–mass spectrometry based metabolite profiling of biological samples is rapidly becoming one of the cornerstones of functional genomics and systems biology. Thus, the technology needs to be available to many laboratories and open exchange of information is required such as those achieved for transcript and protein data. The key‐step in metabolite profiling is the unambiguous identification of metabolites in highly complex metabolite preparations with composite structure. Collections of mass spectra, which comprise frequently observed identified and non‐identified metabolites, represent the most effective means to pool the identification efforts currently performed in many laboratories around the world. Here, we describe a platform for mass spectral and retention time index libraries that will enable this process (MSRI; www.csbdb.mpimp‐golm.mpg.de/gmd.html). This resource should ameliorate many of the problems that each laboratory will face both for the initial establishment of metabolome analysis and for its maintenance at a constant sample throughput.

Metabolomics-assisted breeding: a viable option for crop improvement?

Metabolomics approaches enable the parallel assessment of the levels of a broad range of metabolites and have been documented to have great value in both phenotyping and diagnostic analyses in plants. These tools have recently been turned to evaluation of the natural variance apparent in metabolite composition. Here, we describe exciting progress made in the identification of the genetic determinants of plant chemical composition, focussing on the application of metabolomics strategies and their integration with other high-throughput technologies. Metabolomics represents an important addition to the tools currently employed in genomics-assisted selection for crop improvement.

Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation.

Marine primary productivity is iron (Fe)-limited in vast regions of the contemporary oceans, most notably the high nutrient low chlorophyll (HNLC) regions. Diatoms often form large blooms upon the relief of Fe limitation in HNLC regions despite their prebloom low cell density. Although Fe plays an important role in controlling diatom distribution, the mechanisms of Fe uptake and adaptation to low iron availability are largely unknown. Through a combination of nontargeted transcriptomic and metabolomic approaches, we have explored the biochemical strategies preferred by Phaeo dactylum tricornutum at growth-limiting levels of dissolved Fe. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport, and nitrate assimilation, were down-regulated. Our results show that this retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electrons by mitochondrial alternative oxidase (AOX) and non-photochemical quenching (NPQ), and augmented Fe-independent oxidative stress responses. Iron limitation leads to the elevated expression of at least three gene clusters absent from the Thalassiosira pseudonana genome that encode for components of iron capture and uptake mechanisms.

The Metabolic Response of Heterotrophic Arabidopsis Cells to Oxidative Stress

To cope with oxidative stress, the metabolic network of plant cells must be reconfigured either to bypass damaged enzymes or to support adaptive responses. To characterize the dynamics of metabolic change during oxidative stress, heterotrophic Arabidopsis (Arabidopsis thaliana) cells were treated with menadione and changes in metabolite abundance and 13C-labeling kinetics were quantified in a time series of samples taken over a 6 h period. Oxidative stress had a profound effect on the central metabolic pathways with extensive metabolic inhibition radiating from the tricarboxylic acid cycle and including large sectors of amino acid metabolism. Sequential accumulation of metabolites in specific pathways indicated a subsequent backing up of glycolysis and a diversion of carbon into the oxidative pentose phosphate pathway. Microarray analysis revealed a coordinated transcriptomic response that represents an emergency coping strategy allowing the cell to survive the metabolic hiatus. Rather than attempt to replace inhibited enzymes, transcripts encoding these enzymes are in fact down-regulated while an antioxidant defense response is mounted. In addition, a major switch from anabolic to catabolic metabolism is signaled. Metabolism is also reconfigured to bypass damaged steps (e.g. induction of an external NADH dehydrogenase of the mitochondrial respiratory chain). The overall metabolic response of Arabidopsis cells to oxidative stress is remarkably similar to the superoxide and hydrogen peroxide stimulons of bacteria and yeast (Saccharomyces cerevisiae), suggesting that the stress regulatory and signaling pathways of plants and microbes may share common elements.

The Critical Role of Arabidopsis Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase during Dark-Induced Starvation

In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) and electron-transfer flavoprotein (ETF) are functionally associated, and ETF accepts electrons from at least nine mitochondrial matrix flavoprotein dehydrogenases and transfers them to ubiquinone in the inner mitochondrial membrane. In addition, the mammalian ETF/ETFQO system plays a key role in β-oxidation of fatty acids and catabolism of amino acids and choline. By contrast, nothing is known of the function of ETF and ETFQO in plants. Sequence analysis of the unique Arabidopsis thaliana homologue of ETFQO revealed high similarity to the mammalian ETFQO protein. Moreover, green fluorescent protein cellular localization experiments suggested a mitochondrial location for this protein. RNA gel blot analysis revealed that Arabidopsis ETFQO transcripts accumulated in long-term dark-treated leaves. Analysis of three independent insertional mutants of Arabidopsis ETFQO revealed a dramatic reduction in their ability to withstand extended darkness, resulting in senescence and death within 10 d after transfer, whereas wild-type plants remained viable for at least 15 d. Metabolite profiling of dark-treated leaves of the wild type and mutants revealed a dramatic decline in sugar levels. In contrast with the wild type, the mutants demonstrated a significant accumulation of several amino acids, an intermediate of Leu catabolism, and, strikingly, high-level accumulation of phytanoyl-CoA. These data demonstrate the involvement of a mitochondrial protein, ETFQO, in the catabolism of Leu and potentially of other amino acids in higher plants and also imply a novel role for this protein in the chlorophyll degradation pathway activated during dark-induced senescence and sugar starvation.

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria

This study indicates that Lys catabolism can directly channel electrons to the mitochondrial electron transport chain in addition to sustaining the operation of the tricarboxylic acid cycle under carbon-limiting conditions. The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Mode of Inheritance of Primary Metabolic Traits in Tomato

To evaluate components of fruit metabolic composition, we have previously metabolically phenotyped tomato (Solanum lycopersicum) introgression lines containing segmental substitutions of wild species chromosome in the genetic background of a cultivated variety. Here, we studied the hereditability of the fruit metabolome by analyzing an additional years harvest and evaluating the metabolite profiles of lines heterozygous for the introgression (ILHs), allowing the evaluation of putative quantitative trait locus (QTL) mode of inheritance. These studies revealed that most of the metabolic QTL (174 of 332) were dominantly inherited, with relatively high proportions of additively (61 of 332) or recessively (80 of 332) inherited QTL and a negligible number displaying the characteristics of overdominant inheritance. Comparison of the mode of inheritance of QTL revealed that several metabolite pairs displayed a similar mode of inheritance of QTL at the same chromosomal loci. Evaluation of the association between morphological and metabolic traits in the ILHs revealed that this correlation was far less prominent, due to a reduced variance in the harvest index within this population. These data are discussed in the context of genomics-assisted breeding for crop improvement, with particular focus on the exploitation of wide biodiversity.

Regulatory Features Underlying Pollination-Dependent and -Independent Tomato Fruit Set Revealed by Transcript and Primary Metabolite Profiling

Indole Acetic Acid 9 (IAA9) is a negative auxin response regulator belonging to the Aux/IAA transcription factor gene family whose downregulation triggers fruit set before pollination, thus giving rise to parthenocarpy. In situ hybridization experiments revealed that a tissue-specific gradient of IAA9 expression is established during flower development, the release of which upon pollination triggers the initiation of fruit development. Comparative transcriptome and targeted metabolome analysis uncovered important features of the molecular events underlying pollination-induced and pollination-independent fruit set. Comprehensive transcriptomic profiling identified a high number of genes common to both types of fruit set, among which only a small subset are dependent on IAA9 regulation. The fine-tuning of Aux/IAA and ARF genes and the downregulation of TAG1 and TAGL6 MADS box genes are instrumental in triggering the fruit set program. Auxin and ethylene emerged as the most active signaling hormones involved in the flower-to-fruit transition. However, while these hormones affected only a small number of transcriptional events, dramatic shifts were observed at the metabolic and developmental levels. The activation of photosynthesis and sucrose metabolism-related genes is an integral regulatory component of fruit set process. The combined results allow a far greater comprehension of the regulatory and metabolic events controlling early fruit development both in the presence and absence of pollination/fertilization.