Jedrzej Szymanski

Metabolomic and transcriptomic stress response of Escherichia coli.

Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time‐resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co‐clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition‐dependent associations between metabolites and transcripts. The results confirm and extend existing models about co‐regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis.

Deciphering Transcriptional and Metabolic Networks Associated with Lysine Metabolism during Arabidopsis Seed Development

In order to elucidate transcriptional and metabolic networks associated with lysine (Lys) metabolism, we utilized developing Arabidopsis (Arabidopsis thaliana) seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor; however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the tricarboxylic acid cycle while largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor while suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.

Analysis of short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and light

Although the influence of temperature, particularly cold, on lipid metabolism is well established, previous studies have focused on long-term responses and have largely ignored the influence of other interacting environmental factors. Here, we present a time-resolved analysis of the early responses of the glycerolipidome of Arabidopsis thaliana plants exposed to various temperatures (4, 21 and 32°C) and light intensities (darkness, 75, 150 and 400 μmol m(-2) s(-1)), including selected combinations. Using a UPLC/MS-based lipidomic platform, we reproducibly measured most glycerolipid species reported for Arabidopsis leaves, including the classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). In addition to known lipids, we have identified previously unobserved compounds, such as 36-C PGs and eukaryotic phospholipids containing 16:3 acyl chains. Occurrence of these lipid species implies the action of new biochemical mechanisms. Exposition of Arabidopsis plants to various light and temperature regimes results in two major effects. The first is the dependence of the saturation level of PC and MGDG pools on light intensity, likely arising from light regulation of de novo fatty acid synthesis. The second concerns an immediate decrease in unsaturated species of PG at high-temperature conditions (32°C), which could mark the first stages of adaptation to heat-stress conditions. Observed changes are discussed in the context of current knowledge, and new hypotheses have been formulated concerning the early stages of the plant response to changing light and temperature conditions.

Salt stress and senescence: identification of cross-talk regulatory components

Summary Long-term salinity stress induces senescence, probably through an involvement of hydrogen peroxide (H2O2)-mediated signalling. This study identifies candidate H2O2-responsive cis-regulatory elements governing gene expression during salinity stress-triggered and developmental senescence.

Mining for robust transcriptional and metabolic responses to long‐term salt stress: a case study on the model legume Lotus japonicus

Translational genomics, the use of model species to generate knowledge about biological processes and the functions of genes, offers great promise to biotechnologists. Few studies have sought robust responses of model plants to environmental stresses, such as salinity, by altering the stress dosage or by repeating experiments in consecutive years and/or different seasons. We mined our published and unpublished data on legume salt acclimation for robust system features at the ionomic, transcriptomic and metabolomic levels. We analysed data from the model legume Lotus japonicus, obtained through six independent, long-term, non-lethal salt stress experiments which were carried out over two consecutive years. Best possible controlled greenhouse conditions were applied and two main questions asked: how reproducible are results obtained from physiologically meaningful salinity experiments, and what degree of bias may be expected if conclusions are drawn from less well-repeated sampling? A surprisingly large fraction of the transcriptional and metabolic responses to salt stress were not reproducible between experiments. A core set of robust changes was found that was shared between experiments. Many of these robust responses were qualitatively and quantitatively conserved between different accessions of the same species, indicating that the robust responses may be a sound starting point for translational genomics.

Linking gene expression and membrane lipid composition of Arabidopsis.

This work examines the connection between changes in gene expression and changes in membrane lipid composition in Arabidopsis exposed to different light and temperature conditions. Three transcriptional programs identified are shown to be coordinated with changes in specific membrane lipids, further supported by lipidomic analysis of selected knockout lines. Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and physical properties in the new environment. Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing large-scale coordination between gene expression and changes in glycerolipid levels during the Arabidopsis thaliana response to light and temperature stimuli. Using a multivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations.

Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of natures capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a ‘high-cholesterol’ model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Stability of metabolic correlations under changing environmental conditions in Escherichia coli--a systems approach.

BACKGROUND Biological systems adapt to changing environments by reorganizing their cellular and physiological program with metabolites representing one important response level. Different stresses lead to both conserved and specific responses on the metabolite level which should be reflected in the underlying metabolic network. METHODOLOGY/PRINCIPAL FINDINGS Starting from experimental data obtained by a GC-MS based high-throughput metabolic profiling technology we here develop an approach that: (1) extracts network representations from metabolic condition-dependent data by using pairwise correlations, (2) determines the sets of stable and condition-dependent correlations based on a combination of statistical significance and homogeneity tests, and (3) can identify metabolites related to the stress response, which goes beyond simple observations about the changes of metabolic concentrations. The approach was tested with Escherichia coli as a model organism observed under four different environmental stress conditions (cold stress, heat stress, oxidative stress, lactose diauxie) and control unperturbed conditions. By constructing the stable network component, which displays a scale free topology and small-world characteristics, we demonstrated that: (1) metabolite hubs in this reconstructed correlation networks are significantly enriched for those contained in biochemical networks such as EcoCyc, (2) particular components of the stable network are enriched for functionally related biochemical pathways, and (3) independently of the response scale, based on their importance in the reorganization of the correlation network a set of metabolites can be identified which represent hypothetical candidates for adjusting to a stress-specific response. CONCLUSIONS/SIGNIFICANCE Network-based tools allowed the identification of stress-dependent and general metabolic correlation networks. This correlation-network-based approach does not rely on major changes in concentration to identify metabolites important for stress adaptation, but rather on the changes in network properties with respect to metabolites. This should represent a useful complementary technique in addition to more classical approaches.

Involvement of a gut–retina axis in protection against dietary glycemia-induced age-related macular degeneration

Significance Food is medicine, and diet impacts the risk for and progression of age-related macular degeneration AMD, but we have few clues as to why. We found that wild-type mice fed a high-glycemic-index diet similar in composition to the Western diet developed a disease state that resembles dry AMD. To gain insight into the mechanism, we used LC-MS– and NMR-based metabolomics to discover diet-, metabolic-, and AMD-associated phenotypes. These studies revealed changes in the gut microbiota that altered the production of metabolites that protected against AMD, including serotonin. Changing the diet to a low-glycemic-index diet, even late in life, arrested the development of AMD, offering dietary interventions for AMD. Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut–retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Label‐free deep shotgun proteomics reveals protein dynamics during tomato fruit tissues development

Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.