Regulation of primary metabolism under oxidative stress

Due to their sessile lifestyle plants are affected significantly in their distribution and performance by many abiotic factors. Water supply, temperature and soil quality are most relevant among abiotic stresses and share similar effects on plant growth. At the cellular level, fluctuating light, drought, high and low temperature as well as soil salinity can cause damage to membranes and proteins, and in many cases this is entailed by oxidative stress, resulting from intracellular production of reactive oxygen species. Previous studies have shown significant differences in stress tolerance of the genetic and biochemical model plant Arabidopsis thaliana which were often correlated with the accumulation of soluble carbohydrates and enzyme activities of the central carbohydrate metabolism. Yet, studies also indicated the necessity of analysing subcellular organisation of the whole primary metabolism rather than only the carbohydrate metabolism to be able to draw a representative picture of the complex mechanisms being involved in plant stress response. In the proposed project, we aim to dissect metabolic and signalling effects on primary metabolism caused by oxidative stress at the subcellular level. Our project is set out to elucidate diurnal regulation and compartment interactions contributing to the re-adjustement of redox homeostasis after a disturbance by abiotic stresses. Plants of the Arabidopsis thaliana accession Columbia-0 as well as the gin-2 mutant defective in hexokinase 1 and the S177A complement of its hexose sensing function will be exposed to conditions of light, heat and cold stress. For subcellular analysis of primary metabolism, the non-aqueous fractionation technique will be applied in combination with high- throughput measurement of the metabolome and proteome. The proposed novel combination of the non-aqueous fractionation method with shotgun proteomics, metabolite profiling and refined data processing techniques are intended to present a more precise picture of the plant cell as a system co- ordinating redox and metabolic activities of its subsystems. This shall involve the compartment- specific analysis of low molecular weight ROS scavengers like ascorbate as well as indicators of oxidative damage, e.g. malone dialdehyde and protective molecules like sugars or proline. Mathematical modelling of both diurnal dynamics and subcellular metabolic interactions of leaf primary metabolism shall yield information about bottlenecks for metabolic adaptation to the applied abiotic stress. Beyond the biochemical analysis and comprehensive systems biological characterization of plant-environment interactions and redox challenges, this project intends to contribute an enhanced methodology that allows understanding of the interplay of the different reaction spaces within a plant cell. With a comprehensive analysis of compartment-specific metabolite concentrations during environmental changes we intend to simulate whole cell redox adjustments, thus proving or disproving the concept of a cellular redox state. This should, in turn, burst breeding concepts aimed at improving plant abiotic stress tolerance.

Due to their sessile lifestyle, plants have evolved molecular strategies to cope with fluctuating environmental conditions which affect their ecology and evolution. During abiotic stress exposure, membrane systems and compartments of plant cells might be affected and even irreversibly damaged. Frequently, such a damage is caused by reactive oxygen species (ROS). Thus, acclimation to abiotic stress, e.g. low temperature, high light or drought, plays an essential role for many temperate plant species. Previous studies showed that stress tolerance is frequently accompanied by increased levels of soluble sugars and tight regulation of enzyme activities in the central carbohydrate metabolism. However, evidence has been provided that subcellular localization of other primary metabolites, e.g. amino acids or organic acids, needs to be resolved experimentally to draw an unambiguous picture of the complex process of plant stress response. The aim of our project was to identify stress- induced regulatory strategies of subcellular plant primary metabolism. We applied the technique of non- aqueous fractionation (NAF) in combination with high-throughput proteomics and metabolomics analyses to reveal diurnal dynamics and stress-induced dynamics of subcellular metabolic reprogramming. Our experiments revealed a correlation of the conserved enzyme Hexokinase 1 with photorespiratory activity which has not been reported before. In particular, reprogramming of cytosolic and vacuolar hexose metabolism was observed to significantly affect the metabolic homeostasis of chloroplasts and mitochondria. Further, applying a mathematical modelling approach we identified vacuolar sucrose cleavage to stabilize plastidial metabolism and photosynthesis during environmental fluctuations. In summary, our project provided evidence for the necessity to combine experimental analysis of subcellular plant metabolism with mathematical modelling and multivariate statistics to identify key players of metabolic reprogramming involved in plant stress response and adaption.