Eco-Metabolomics and Metabolic Modeling: Making the Leap From Model Systems in the Lab to Native Populations in the Field

Experimental high-throughput analysis of molecular networks is a central approach to characterize the adaptation of plant metabolism to the environment. However, recent studies have demonstrated that it is hardly possible to predict in situ metabolic phenotypes from experiments under controlled conditions, such as growth chambers or greenhouses. This is particularly due to the high molecular variance of in situ samples induced by environmental fluctuations. An approach of functional metabolome interpretation of field samples would be desirable in order to be able to identify and trace back the impact of environmental changes on plant metabolism. To test the applicability of metabolomics studies for a characterization of plant populations in the field, we have identified and analyzed in situ samples of nearby grown natural populations of Arabidopsis thaliana in Austria. A. thaliana is the primary molecular biological model system in plant biology with one of the best functionally annotated genomes representing a reference system for all other plant genome projects. The genomes of these novel natural populations were sequenced and phylogenetically compared to a comprehensive genome database of A. thaliana ecotypes. Experimental results on primary and secondary metabolite profiling and genotypic variation were functionally integrated by a data mining strategy, which combines statistical output of metabolomics data with genome-derived biochemical pathway reconstruction and metabolic modeling. Correlations of biochemical model predictions and population-specific genetic variation indicated varying strategies of metabolic regulation on a population level which enabled the direct comparison, differentiation, and prediction of metabolic adaptation of the same species to different habitats. These differences were most pronounced at organic and amino acid metabolism as well as at the interface of primary and secondary metabolism and allowed for the direct classification of population-specific metabolic phenotypes within geographically contiguous sampling sites

Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.FPA and co-workers are funded by the Spanish MICINN-FEDER (projects AT2009-0038 and AGL2011-27996) and the European Commission (ROOTOPOWER Contract # 289365). TR and FPA were jointly funded by the Spanish–Austrian bilateral project AT2009-0038. AA was supported by post-doctoral fellowships from the Fundación Séneca (Comunidad Autónoma de la Región de Murcia) and the FWF (Austrian Science Fund), and currently by the JAE DOC Programme

Solving the differential biochemical Jacobian from metabolomics covariance data.

High-throughput molecular analysis has become an integral part in organismal systems biology. In contrast, due to a missing systematic linkage of the data with functional and predictive theoretical models of the underlying metabolic network the understanding of the resulting complex data sets is lacking far behind. Here, we present a biomathematical method addressing this problem by using metabolomics data for the inverse calculation of a biochemical Jacobian matrix, thereby linking computer-based genome-scale metabolic reconstruction and in vivo metabolic dynamics. The incongruity of metabolome coverage by typical metabolite profiling approaches and genome-scale metabolic reconstruction was solved by the design of superpathways to define a metabolic interaction matrix. A differential biochemical Jacobian was calculated using an approach which links this metabolic interaction matrix and the covariance of metabolomics data satisfying a Lyapunov equation. The predictions of the differential Jacobian from real metabolomic data were found to be correct by testing the corresponding enzymatic activities. Moreover it is demonstrated that the predictions of the biochemical Jacobian matrix allow for the design of parameter optimization strategies for ODE-based kinetic models of the system. The presented concept combines dynamic modelling strategies with large-scale steady state profiling approaches without the explicit knowledge of individual kinetic parameters. In summary, the presented strategy allows for the identification of regulatory key processes in the biochemical network directly from metabolomics data and is a fundamental achievement for the functional interpretation of metabolomics data

Primary Metabolism, Phenylpropanoids and Antioxidant Pathways Are Regulated in Potato as a Response to Potato virus Y Infection.

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus-plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed

Altitudinal patterns of diversity and functional traits of metabolically active microorganisms in stream biofilms

Resources structure ecological communities and potentially link biodiversity to energy flow. It is commonly believed that functional traits (generalists versus specialists) involved in the exploitation of resources depend on resource availability and environmental fluctuations. The longitudinal nature of stream ecosystems provides changing resources to stream biota with yet unknown effects on microbial functional traits and community structure. We investigated the impact of autochthonous (algal extract) and allochthonous (spruce extract) resources, as they change along alpine streams from above to below the treeline, on microbial diversity, community composition and functions of benthic biofilms. Combining bromodeoxyuridine labelling and 454 pyrosequencing, we showed that diversity was lower upstream than downstream of the treeline and that community composition changed along the altitudinal gradient. We also found that, especially for allochthonous resources, specialisation by biofilm bacteria increased along that same gradient. Our results suggest that in streams below the treeline biofilm diversity, specialisation and functioning are associated with increasing niche differentiation as potentially modulated by divers allochthonous and autochthonous constituents contributing to resources. These findings expand our current understanding on biofilm structure and function in alpine streams

Male Sterility in Maize after Transient Heat Stress during the Tetrad Stage of Pollen Development

Shifts in the duration and intensity of ambient temperature impair plant development and reproduction, particularly male gametogenesis. Stress exposure causes meiotic defects or premature spore abortion in male reproductive organs, leading to male sterility. However, little is known about the mechanisms underlying stress and male sterility. To elucidate these mechanisms, we imposed a moderate transient heat stress on maize (Zea mays) plants at the tetrad stage of pollen development. After completion of pollen development at optimal conditions, stress responses were assessed in mature pollen. Transient heat stress resulted in reduced starch content, decreased enzymatic activity, and reduced pollen germination, resulting in sterility. A transcriptomic comparison pointed toward misregulation of starch, lipid, and energy biosynthesis-related genes. Metabolomic studies showed an increase of Suc and its monosaccharide components, as well as a reduction in pyruvate. Lipidomic analysis showed increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids. In contrast, the majority of genes involved in developmental processes such as those required for auxin and unfolded protein responses, signaling, and cell wall biosynthesis remained unaltered. It is noteworthy that changes in the regulation of transcriptional and metabolic pathway genes, as well as heat stress proteins, remained altered even though pollen could recover during further development at optimal conditions. In conclusion, our findings demonstrate that a short moderate heat stress during the highly susceptible tetrad stage strongly affects basic metabolic pathways and thus generates germination-defective pollen, ultimately leading to severe yield losses in maize