Artemisinin-based therapies are the only effective treatment for malaria, the most devastating disease in human history. To meet the growing demand for artemisinin and make it accessible to the ...poorest, an inexpensive and rapidly scalable production platform is urgently needed. Here we have developed a new synthetic biology approach, combinatorial supertransformation of transplastomic recipient lines (COSTREL), and applied it to introduce the complete pathway for artemisinic acid, the precursor of artemisinin, into the high-biomass crop tobacco. We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome. The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway. By screening large populations of COSTREL lines, we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass. Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output.
Motivation: Typical GC-MS-based metabolite profiling experiments may comprise hundreds of chromatogram files, which each contain up to 1000 mass spectral tags (MSTs). MSTs are the characteristic ...patterns of ∼25–250 fragment ions and respective isotopomers, which are generated after gas chromatography (GC) by electron impact ionization (EI) of the separated chemical molecules. These fragment ions are subsequently detected by time-of-flight (TOF) mass spectrometry (MS). MSTs of profiling experiments are typically reported as a list of ions, which are characterized by mass, chromatographic retention index (RI) or retention time (RT), and arbitrary abundance. The first two parameters allow the identification, the later the quantification of the represented chemical compounds. Many software tools have been reported for the pre-processing, the so-called curve resolution and deconvolution, of GC-(EI-TOF)-MS files. Pre-processing tools generate numerical data matrices, which contain all aligned MSTs and samples of an experiment. This process, however, is error prone mainly due to (i) the imprecise RI or RT alignment of MSTs and (ii) the high complexity of biological samples. This complexity causes co-elution of compounds and as a consequence non-selective, in other words impure MSTs. The selection and validation of optimal fragment ions for the specific and selective quantification of simultaneously eluting compounds is, therefore, mandatory. Currently validation is performed in most laboratories under human supervision. So far no software tool supports the non-targeted and user-independent quality assessment of the data matrices prior to statistical analysis. TagFinder may fill this gap. Strategy: TagFinder facilitates the analysis of all fragment ions, which are observed in GC-(EI-TOF)-MS profiling experiments. The non-targeted approach allows the discovery of novel and unexpected compounds. In addition, mass isotopomer resolution is maintained by TagFinder processing. This feature is essential for metabolic flux analyses and highly useful, but not required for metabolite profiling. Whenever possible, TagFinder gives precedence to chemical means of standardization, for example, the use of internal reference compounds for retention time calibration or quantitative standardization. In addition, external standardization is supported for both compound identification and calibration. The workflow of TagFinder comprises, (i) the import of fragment ion data, namely mass, time and arbitrary abundance (intensity), from a chromatography file interchange format or from peak lists provided by other chromatogram pre-processing software, (ii) the annotation of sample information and grouping of samples into classes, (iii) the RI calculation, (iv) the binning of observed fragment ions of equal mass from different chromatograms into RI windows, (v) the combination of these bins, so-called mass tags, into time groups of co-eluting fragment ions, (vi) the test of time groups for intensity correlated mass tags, (vii) the data matrix generation and (viii) the extraction of selective mass tags supported by compound identification. Thus, TagFinder supports both non-targeted fingerprinting analyses and metabolite targeted profiling. Availability: Exemplary TagFinder workspaces and test data sets are made available upon request to the contact authors. TagFinder is made freely available for academic use from http://www-en.mpimp-golm.mpg.de/03-research/researchGroups/01-dept1/Root_Metabolism/smp/TagFinder/index.html Contact: Kopka@mpimp-golm.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online and within the TagFinder download from the above URL.
Cold acclimation (CA) leads to increased plant freezing tolerance during exposure to low, non-freezing temperatures as a result of many physiological, biochemical and molecular changes that have been ...extensively investigated. In addition, many plant species, such as Arabidopsis thaliana, respond to a subsequent exposure to mild, non-damaging freezing temperatures with an additional increase in freezing tolerance referred to as sub-zero acclimation (SZA). There is comparatively little information available about the molecular basis of SZA. However, previous transcriptomic studies indicated that cell wall modification may play an important role during SZA. Here we show that CA and SZA are accompanied by extensive changes in cell wall amount, composition and structure. While CA leads to a significant increase in cell wall amount, the relative proportions of pectin, hemicellulose and cellulose remained unaltered during both CA and SZA. However, both treatments resulted in more subtle changes in structure as determined by infrared spectroscopy and monosaccharide composition as determined by gas chromatography-mass spectrometry. These differences could be related through a proteomic approach to the accumulation of cell wall modifying enzymes such as pectin methylesterases, pectin methylesterase inhibitors and xyloglucan endotransglucosylases/hydrolases in the extracellular matrix.
Ribosome biogenesis is tightly associated to plant metabolism due to the usage of ribosomes in the synthesis of proteins necessary to drive metabolic pathways. Given the central role of ribosome ...biogenesis in cell physiology, it is important to characterize the impact of different components involved in this process on plant metabolism. Double mutants of the Arabidopsis thaliana cytosolic 60S maturation factors REIL1 and REIL2 do not resume growth after shift to moderate 10 Formula: see text chilling conditions. To gain mechanistic insights into the metabolic effects of this ribosome biogenesis defect on metabolism, we developed TC-iReMet2, a constraint-based modelling approach that integrates relative metabolomics and transcriptomics time-course data to predict differential fluxes on a genome-scale level. We employed TC-iReMet2 with metabolomics and transcriptomics data from the Arabidopsis Columbia 0 wild type and the reil1-1 reil2-1 double mutant before and after cold shift. We identified reactions and pathways that are highly altered in a mutant relative to the wild type. These pathways include the Calvin-Benson cycle, photorespiration, gluconeogenesis, and glycolysis. Our findings also indicated differential NAD(P)/NAD(P)H ratios after cold shift. TC-iReMet2 allows for mechanistic hypothesis generation and interpretation of system biology experiments related to metabolic fluxes on a genome-scale level.
Many organisms form elaborate mineralized structures, constituted of highly organized arrangements of crystals and organic macromolecules. The localization of crystals within these structures is ...presumably determined by the interaction of nucleating macromolecules with the mineral phase. Here we show that, preceding nucleation, a specific interaction between soluble organic molecules and an organic backbone structure directs mineral components to specific sites. This strategy underlies the formation of coccoliths, which are highly ordered arrangements of calcite crystals produced by marine microalgae. On combining the insoluble organic coccolith scaffold with coccolith-associated soluble macromolecules in vitro, we found a massive accretion of calcium ions at the sites where the crystals form in vivo. The in vitro process exhibits profound similarities to the initial stages of coccolith biogenesis in vivo.
Plants need to adapt to fluctuating temperatures throughout their lifetime. Previous research showed that Arabidopsis memorizes a first cold stress (priming) and improves its primed freezing ...tolerance further when subjected to a second similar stress after a lag phase. This study investigates primary metabolomic and transcriptomic changes during early cold priming or triggering after 3 days at 4°C interrupted by a memory phase. DREB1 family transcription factors DREB1C/CBF2, DREB1D/CBF4, DREB1E/DDF2, and DREB1F/DDF1 were strongly significantly induced throughout the entire triggering. During triggering, genes encoding Late Embryogenesis Abundant (LEA), antifreeze proteins or detoxifiers of reactive oxygen species (ROS) were higher expressed compared with priming. Examples of early triggering responders were xyloglucan endotransglucosylase/hydrolase genes encoding proteins involved in cell wall remodeling, while late responders were identified to act in fine‐tuning the stress response and developmental regulation. Induction of non‐typical members of the DREB subfamily of ERF/AP2 transcription factors, the relatively small number of induced CBF regulon genes and a slower accumulation of selected cold stress associated metabolites indicate that a cold triggering stimulus might be sensed as milder stress in plants compared with priming. Further, strong induction of CBF4 throughout triggering suggests a unique function of this gene for the response to alternating temperatures.
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 approximately 400 experimental samples per week per machine.
Metabolomics of temperature stress Guy, Charles; Kaplan, Fatma; Kopka, Joachim ...
Physiologia plantarum,
February 2008, Letnik:
132, Številka:
2
Journal Article
Recenzirano
Plants possess inducible tolerance mechanisms that extend the temperature range for survival during acute temperature stress. The inducible mechanisms of cold acclimation and acquired thermotolerance ...involve highly complex processes. These include perception and signal transduction of non-optimal temperatures or their physical consequences on cellular components that program extensive modification of the transcriptome, proteome, metabolome and composition and physical structure of the cytoplasm, membranes and cell walls. Therefore, a systems biology approach will be necessary to advance the understanding of plant stress responses and tolerance mechanisms. One promise of systems biology is that it will greatly enhance our understanding of individual and collective functions and thereby provide a more holistic view of plant stress responses. Past studies have found that several metabolites that could functionally contribute to induced stress tolerance have been associated with stress responses. Recent metabolite-profiling studies have refocused attention on these and other potentially important components found in the 'temperature-stress metabolome'. These metabolomic studies have demonstrated that active reconfiguration of the metabolome is regulated in part by changes in gene expression initiated by temperature-stress-activated signaling and stress-related transcription factors. One aspect of metabolism that is consistent across all of the temperature-stress metabolomic studies to date is the prominent role of central carbohydrate metabolism, which seems to be a major feature of the reprogramming of the metabolome during temperature stress. Future metabolomic studies of plant temperature-stress responses should reveal additional metabolic pathways that have important functions in temperature-stress tolerance mechanisms.
Gas chromatography coupled to mass spectrometry (GC-MS) is one of the most widespread routine technologies applied to the large scale screening and discovery of novel metabolic biomarkers. However, ...currently the majority of mass spectral tags (MSTs) remains unidentified due to the lack of authenticated pure reference substances required for compound identification by GC-MS. Here, we accessed the information on reference compounds stored in the Golm Metabolome Database (GMD) to apply supervised machine learning approaches to the classification and identification of unidentified MSTs without relying on library searches. Non-annotated MSTs with mass spectral and retention index (RI) information together with data of already identified metabolites and reference substances have been archived in the GMD. Structural feature extraction was applied to sub-divide the metabolite space contained in the GMD and to define the prediction target classes. Decision tree (DT)-based prediction of the most frequent substructures based on mass spectral features and RI information is demonstrated to result in highly sensitive and specific detections of sub-structures contained in the compounds. The underlying set of DTs can be inspected by the user and are made available for batch processing via SOAP (Simple Object Access Protocol)-based web services. The GMD mass spectral library with the integrated DTs is freely accessible for non-commercial use at http://gmd.mpimp-golm.mpg.de/. All matching and structure search functionalities are available as SOAP-based web services. A XML + HTTP interface, which follows Representational State Transfer (REST) principles, facilitates read-only access to data base entities.
Plants dedicate a high amount of energy and resources to the production of ribosomes. Historically, these multi-protein ribosome complexes have been considered static protein synthesis machines that ...are not subject to extensive regulation but only read mRNA and produce polypeptides accordingly. New and increasing evidence across various model organisms demonstrated the heterogeneous nature of ribosomes. This heterogeneity can constitute specialized ribosomes that regulate mRNA translation and control protein synthesis. A prominent example of ribosome heterogeneity is seen in the model plant, Arabidopsis thaliana, which, due to genome duplications, has multiple paralogs of each ribosomal protein (RP) gene. We support the notion of plant evolution directing high RP paralog divergence toward functional heterogeneity, underpinned in part by a vast resource of ribosome mutants that suggest specialization extends beyond the pleiotropic effects of single structural RPs or RP paralogs. Thus, Arabidopsis is a highly suitable model to study this phenomenon. Arabidopsis enables reverse genetics approaches that could provide evidence of ribosome specialization. In this review, we critically assess evidence of plant ribosome specialization and highlight steps along ribosome biogenesis in which heterogeneity may arise, filling the knowledge gaps in plant science by providing advanced insights from the human or yeast fields. We propose a data analysis pipeline that infers the heterogeneity of ribosome complexes and deviations from canonical structural compositions linked to stress events. This analysis pipeline can be extrapolated and enhanced by combination with other high-throughput methodologies, such as proteomics. Technologies, such as kinetic mass spectrometry and ribosome profiling, will be necessary to resolve the temporal and spatial aspects of translational regulation while the functional features of ribosomal subpopulations will become clear with the combination of reverse genetics and systems biology approaches.
PDF
