Metabolic support by macrophages sustains colonic epithelial homeostasis.

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

Metabolome plasticity in 241 Arabidopsis thaliana accessions reveals evolutionary cold adaptation processes.

Acclimation and adaptation of metabolism to a changing environment are key processes for plant survival and reproductive success. In the present study, 241 natural accessions of Arabidopsis (Arabidopsis thaliana) were grown under two different temperature regimes, 16 °C and 6 °C, and growth parameters were recorded, together with metabolite profiles, to investigate the natural genome × environment effects on metabolome variation. The plasticity of metabolism, which was captured by metabolic distance measures, varied considerably between accessions. Both relative growth rates and metabolic distances were predictable by the underlying natural genetic variation of accessions. Applying machine learning methods, climatic variables of the original growth habitats were tested for their predictive power of natural metabolic variation among accessions. We found specifically habitat temperature during the first quarter of the year to be the best predictor of the plasticity of primary metabolism, indicating habitat temperature as the causal driver of evolutionary cold adaptation processes. Analyses of epigenome- and genome-wide associations revealed accession-specific differential DNA-methylation levels as potentially linked to the metabolome and identified FUMARASE2 as strongly associated with cold adaptation in Arabidopsis accessions. These findings were supported by calculations of the biochemical Jacobian matrix based on variance and covariance of metabolomics data, which revealed that growth under low temperatures most substantially affects the accession-specific plasticity of fumarate and sugar metabolism. Our findings indicate that the plasticity of metabolic regulation is predictable from the genome and epigenome and driven evolutionarily by Arabidopsis growth habitats.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Dynamics of Plant Metabolism during Cold Acclimation.

Plants have evolved strategies to tightly regulate metabolism during acclimation to a changing environment. Low temperature significantly constrains distribution, growth and yield of many temperate plant species. Exposing plants to low but non-freezing temperature induces a multigenic processes termed cold acclimation, which eventually results in an increased freezing tolerance. Cold acclimation comprises reprogramming of the transcriptome, proteome and metabolome and affects communication and signaling between subcellular organelles. Carbohydrates play a central role in this metabolic reprogramming. This review summarizes current knowledge about the role of carbohydrate metabolism in plant cold acclimation with a focus on subcellular metabolic reprogramming, its thermodynamic constraints under low temperature and mathematical modelling of metabolism.

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3ß-MCL-1 Axis.

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3ß (GSK3ß) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3ß axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3ß.

Vacuolar sucrose cleavage prevents limitation of cytosolic carbohydrate metabolism and stabilizes photosynthesis under abiotic stress.

Stabilization of central carbohydrate metabolism plays a key role in plant stress response. Carbohydrates are substrate for numerous metabolic and stress-responsive reactions and have been shown to be involved in diverse signalling processes on a whole-plant level. Regulation of enzymatic sucrose synthesis and degradation is well-known to be central to many stress-related processes as it significantly impacts stress tolerance. Leaf sucrose metabolism involves sucrose cleavage by invertases and ATP-consuming resynthesis catalysed by hexokinase and sucrose phosphate synthase. These reactions establish a metabolic cycle. To study the physiological role of sucrose cycling, a kinetic model was developed to simulate dynamics of subcellular sugar concentrations in Arabidopsis thaliana under combined cold and high-light stress. Model simulation revealed that subcellular reprogramming of invertase-driven sucrose cleavage varies substantially between natural accessions of Arabidopsis which differ in their cold tolerance levels. A stress-induced shift of sucrose cleavage from the cytosol into the vacuole could only be observed for the tolerant accession while the susceptible accession increased the cytosolic proportion of sucrose cleavage. Under stress, reduction in vacuolar invertase activity significantly affected maximum quantum yield of photosystem II and CO2 assimilation rates. While wild-type plants circumvented a limitation of sucrose cleavage by increasing vacuolar invertase activity, mutant plants were not able to compensate their deficiency of vacuolar by cytosolic activity. Consequently, the capacity for cytosolic hexose generation was lower than for enzymatic hexose phosphorylation suggesting a role of vacuolar invertase activity in preventing a limitation in cytosolic hexose metabolism under stress. ENZYMES: Invertase, EC 3.2.1.26; Hexokinase, EC 2.7.1.1.

Mathematical Modeling Approaches in Plant Metabolomics.

The experimental analysis of a plant metabolome typically results in a comprehensive and multidimensional data set. To interpret metabolomics data in the context of biochemical regulation and environmental fluctuation, various approaches of mathematical modeling have been developed and have proven useful. In this chapter, a general introduction to mathematical modeling is presented and discussed in context of plant metabolism. A particular focus is laid on the suitability of mathematical approaches to functionally integrate plant metabolomics data in a metabolic network and combine it with other biochemical or physiological parameters.

System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism.

Grapevine is a fruit crop with worldwide economic importance. The grape berry undergoes complex biochemical changes from fruit set until ripening. This ripening process and production processes define the wine quality. Thus, a thorough understanding of berry ripening is crucial for the prediction of wine quality. For a systemic analysis of grape berry development we applied mass spectrometry based platforms to analyse the metabolome and proteome of Early Campbell at 12 stages covering major developmental phases. Primary metabolites involved in central carbon metabolism, such as sugars, organic acids and amino acids together with various bioactive secondary metabolites like flavonols, flavan-3-ols and anthocyanins were annotated and quantified. At the same time, the proteomic analysis revealed the protein dynamics of the developing grape berries. Multivariate statistical analysis of the integrated metabolomic and proteomic dataset revealed the growth trajectory and corresponding metabolites and proteins contributing most to the specific developmental process. K-means clustering analysis revealed 12 highly specific clusters of co-regulated metabolites and proteins. Granger causality network analysis allowed for the identification of time-shift correlations between metabolite-metabolite, protein- protein and protein-metabolite pairs which is especially interesting for the understanding of developmental processes. The integration of metabolite and protein dynamics with their corresponding biochemical pathways revealed an energy-linked metabolism before veraison with high abundances of amino acids and accumulation of organic acids, followed by protein and secondary metabolite synthesis. Anthocyanins were strongly accumulated after veraison whereas other flavonoids were in higher abundance at early developmental stages and decreased during the grape berry developmental processes. A comparison of the anthocyanin profile of Early Campbell to other cultivars revealed similarities to Concord grape and indicates the strong effect of genetic background on metabolic partitioning in primary and secondary metabolism.

System level analysis of cacao seed ripening reveals a sequential interplay of primary and secondary metabolism leading to polyphenol accumulation and preparation of stress resistance.

Theobroma cacao and its popular product, chocolate, are attracting attention due to potential health benefits including antioxidative effects by polyphenols, anti-depressant effects by high serotonin levels, inhibition of platelet aggregation and prevention of obesity-dependent insulin resistance. The development of cacao seeds during fruit ripening is the most crucial process for the accumulation of these compounds. In this study, we analyzed the primary and the secondary metabolome as well as the proteome during Theobroma cacao cv. Forastero seed development by applying an integrative extraction protocol. The combination of multivariate statistics and mathematical modelling revealed a complex consecutive coordination of primary and secondary metabolism and corresponding pathways. Tricarboxylic acid (TCA) cycle and aromatic amino acid metabolism dominated during the early developmental stages (stages 1 and 2; cell division and expansion phase). This was accompanied with a significant shift of proteins from phenylpropanoid metabolism to flavonoid biosynthesis. At stage 3 (reserve accumulation phase), metabolism of sucrose switched from hydrolysis into raffinose synthesis. Lipids as well as proteins involved in lipid metabolism increased whereas amino acids and N-phenylpropenoyl amino acids decreased. Purine alkaloids, polyphenols, and raffinose as well as proteins involved in abiotic and biotic stress accumulated at stage 4 (maturation phase) endowing cacao seeds the characteristic astringent taste and resistance to stress. In summary, metabolic key points of cacao seed development comprise the sequential coordination of primary metabolites, phenylpropanoid, N-phenylpropenoyl amino acid, serotonin, lipid and polyphenol metabolism thereby covering the major compound classes involved in cacao aroma and health benefits.

A Strategy for Functional Interpretation of Metabolomic Time Series Data in Context of Metabolic Network Information.

The functional connection of experimental metabolic time series data with biochemical network information is an important, yet complex, issue in systems biology. Frequently, experimental analysis of diurnal, circadian, or developmental dynamics of metabolism results in a comprehensive and multidimensional data matrix comprising information about metabolite concentrations, protein levels, and/or enzyme activities. While, irrespective of the type of organism, the experimental high-throughput analysis of the transcriptome, proteome, and metabolome has become a common part of many systems biological studies, functional data integration in a biochemical and physiological context is still challenging. Here, an approach is presented which addresses the functional connection of experimental time series data with biochemical network information which can be inferred, for example, from a metabolic network reconstruction. Based on a time-continuous and variance-weighted regression analysis of experimental data, metabolic functions, i.e., first-order derivatives of metabolite concentrations, were related to time-dependent changes in other biochemically relevant metabolic functions, i.e., second-order derivatives of metabolite concentrations. This finally revealed time points of perturbed dependencies in metabolic functions indicating a modified biochemical interaction. The approach was validated using previously published experimental data on a diurnal time course of metabolite levels, enzyme activities, and metabolic flux simulations. To support and ease the presented approach of functional time series analysis, a graphical user interface including a test data set and a manual is provided which can be run within the numerical software environment Matlab®.