The pathways leading to the indolic amino acid tryptophan are of critical importance because tryptophan is required in animal and human diets and because indolic compounds are used as intercellular signals in plants and animals. In addition, indolic pathways reside at the interface of primary and secondary metabolism and function as a node in dynamic metabolic processes that may be predictive of the metabolic status of individual cell types. The objective of this project is to demonstrate the potential of metabolite quantification or profiling methods (metabolomics) to provide insight at the single cell level into the cellular and metabolic consequences of disrupting genes involved in tryptophan biosynthesis. This approach will answer important questions about differences in metabolism between cells that are in close proximity to one another, but which differ in their origin, function or ultimate fate. The project will take advantage of approaches that have been effective when applied to cell-specific transcript profiling, namely sorting of cells with fluorescent protein-tagged fusion proteins in specific cell types, and will apply them to targeted metabolite profiling on a submicro-scale. Tryptophan biosynthesis is an excellent metabolic pathway for such studies not only because the pathway exists at the interface between primary and secondary metabolism, but also because increased tryptophan levels are a potentially valuable crop plant trait and a target for metabolic engineering for animal and human health. A project website (www.auxin.org) will detail the specific objectives and progress of the project, including metabolite profiling data. Mutant lines will be deposited at the ABRC.
Broader Impacts: The major objective of this project is to advance the methodology of metabolomics (metabolite quantification) at the tissue and cellular levels to the point that it is routinely available to the broader plant community as part of the functional genomics toolbox. The PIs will develop a workshop to disseminate metabolomics methods to the broader scientific community. This project will train postdoctoral fellows, who will be well-positioned to continue the development and application of metabolomics as a genomics tool. The project will interface with on-going activities to recruit and train members of underrepresented groups.
The proposed activities meet the goals of the 2010 Project by addressing a recognized need to focus on metabolite profiling in order to characterize the localizome, that is, information about gene and protein expression patterns and metabolite concentrations and flux at the organ, tissue, cellular and/or subcellular levels in the living plant. As noted in the 2010 Project mid-course assessment, knowledge of small molecule biology in Arabidopsis is not only fundamental for a holistic understanding of plant biology, but also in the context of plants as a source of food for both humans and animals as well as sources of pharmaceuticals. As an essential amino acid, tryptophan biosynthesis is broadly relevant, and the approaches developed will allow the study of any metabolite network in plants.
