Participants:
Jerry Cohen,
lead PI
Jennifer Normanly
John Celenza
(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.
Cellular and
Subcellular Resolution of the
Tryptophan-Related Pathways