NSFCellular and Subcellular Resolution of the Tryptophan-Related Pathways
                (Funded by the  NSF 2010 Project   -  awards 0725149, 0725192, and 0724970)


Jerry Cohen, lead PIUMinn
University of Minnesota
Dept of Horticultural Science

252 Alderman Hall
1970 Folwell Avenue
St. Paul, MN 55108

Jennifer Normanlyumass
University of Massachusetts, Amherst
Dept of Biochemistry and Molecular Biology
710 N. Pleasant St. LGRT
Amherst, MA 01003

John Celenza
Boston University
Dept of Biology
5 Cummington Street
Boston, MA 02215

Publications resulting from this project

Target genes for 2010

Project Summary

This is a collaborative proposal between three PIs. The objective of this 2010 Project is to develop targeted, quantitative metabolomics 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. Quantitative targeted metabolomics of aromatic and indolic metabolites will be carried out on Arabidopsis lines with genetic and environmental (light) 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 GFP-tagged fusion proteins in specific cell types, will be applied to targeted metabolomics. Similarly, GFP-tagged chloroplast proteins will be used to develop facile validation methods for subfractionation of cells. This project is uniquely poised to make headway with regard to quantification of the target metabolites by leveraging the availability of stable-isotope labeled compounds from a current NSF Plant Genome proteomics project. There are three specific aims in this collaborative project, 1) the development of broadly applicable and accessible methods for quantitative targeted metabolomics, 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 metabolite quantification 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.

Broader Impacts Metabolomics has the potential to provide a physiological snapshot of an organelle, cell, tissue or whole organism, and as such should be a ubiquitous component of the functional genomics toolbox. The major objective of this project is to advance the methodology of metabolite quantification at the tissue, cellular and subcellular levels to the point that it is routinely available to the broader plant community. All the PIs will participate in instructional workshops aimed at bringing metabolomics methods to the plant community. This project will train graduate students, postdoctoral fellows and undergraduates, who will be well-positioned to participate in the continued development and application of metabolomics as a genomics tool. All the PIs are actively involved in their respective institutions’ activities to recruit and train members of underrepresented groups.