| Low abundance phosphorylated sugars such as trehalose-6-phosphate (T6P) are essential regulatory compounds in insects and plants since absence of T6P is lethal. In plants as well as in other organisms, the mode of action of T6P is unknown, yet T6P?s essential control over carbon utilization likely underlies its multiple effects. Analytical tools for comprehensive and high throughput profiling of low abundance phosphorylated sugar intermediates are lacking, however, and should be developed. In this project target processes of T6P will be studied by combining new metabolite profiling techniques with genetics using the model plant Arabidopsis. Capillary electrophoresis (CE) is a very efficient separation technique particularly useful for profiling of small ionic compounds. The applicability of CE for metabolomics purposes, however, is still limited due to an inherently moderate concentration sensitivity. The combination of CE with advanced affinity extraction methodologies would provide pre-concentration of target analytes yielding a significant improvement of both selectivity and sensitivity. We will design an affinity solid-phase extraction?capillary electrophoresis (ASPE?CE) platform for the highly specific determination of phosphorylated sugars in plants. To this end, micro-columns with metal-affinity phases will be coupled to CE in an on-line fashion allowing fast and automated analysis. The ASPE?CE system will be combined with mass spectrometric (MS) detection in order to characterise and identify (unknown) separated species. Application of ASPE?CE to Arabidopsis mutants and Recombinant Inbred Lines (RILs) altered in T6P metabolism will reveal processes that control T6P steady state and thus affect carbon utilization. Mutants and RILs of Arabidopsis that no longer respond to high endogenous T6P will uncover targets of T6P regulation. Determination of T6P levels in relation to levels of other phosphorylated intermediates, such as phosphorylated fructose or glucose, will reveal network relationships between these compounds. Profiling of phosphorylated intermediates will also be applied in a general genetical metabolomics approach to reveal loci controlling networks of these compounds. The gene products of these loci are also potential targets of T6P regulation. |
