The objective of this NSF2010 funded project is to develop targeted metabolite profiling methods with cellular and subcellular resolution. The project will focus upon a metabolic pathway that is at the interface of primary and secondary metabolism and functions as a node in overlapping, dynamic metabolic processes. Specifically, this project will focus upon determining at the cellular and subcellular level, the metabolic consequences of genetic disruption and dysregulation of tryptophan (Trp) biosynthesis. Targeted metabolite profiling of aromatic and indolic metabolites will be carried out on Arabidopsis lines with genetic and environmental perturbations to Trp biosynthesis. Trp biosynthesis is an exemplary metabolic pathway not only because of its location at the interface between primary and secondary metabolism, but also because an increase in its levels is a potentially valuable crop plant trait relevant to metabolic engineering for animal and human health. Trp synthesis is well supported genetically in Arabidopsis, with each step in the biosynthetic pathway biochemically annotated; however, there are a number of fundamental questions about metabolic networks in general and Trp metabolism in particular that need to be addressed. Specifically, what are the functions of the redundant TRP genes in primary and secondary metabolism? For example, Trp synthase alpha (TSA)1 and TS beta (TSB)1 are nominally involved in Trp synthesis, but what are the functions of the additional TSA and TSB homologs? On a broader scale, the impact of these genes on other metabolic pathways is a certainty; however, quantification of this impact has yet to be addressed experimentally at the cellular and subcellular level. The approaches applied to cell-specific transcript profiling, namely cell sorting of fluorescent protein-tagged fusion proteins in specific cell types, will be applied to targeted metabolite profiling. Similarly, GFP-tagged chloroplast proteins will be used to develop facile validation methods for subfractionation of cells. There are three specific aims in this collaborative project, 1) the development of broadly applicable and accessible methods for targeted metabolite profiling, 2a) the development of methods to achieve cellular and subcellular resolution in metabolite profiling that use commonly available Arabidopsis lines, with cell type-specific fluorescent-tagged proteins for cell sorting, and subcellular-specific fluorescent-tagged proteins for facile assessment of subcellular fractionation purity, 2b) targeted profiling of aromatic and indolic metabolites in selected Trp-dysregulated mutant lines (subjected to high and low light) at three levels of resolution (organ, cellular and subcellular), and 3) the creation and analysis gene expression reporters for selected Trp metabolism genes and functional characterization of previously uncharacterized Trp biosynthetic genes.