Proteomics of Arabidopsis thaliana and Poplar tree plastids: Understanding carbon allocation through the phenylpropanoid pathway
EMSL Project ID
35092
Abstract
In this proposal, we wish to investigate, identify, and establish the factors affecting phenylalanine (Phe) biosynthesis in plastids of vascular plants, together with that of its transport to the cytosol and its subsequent metabolism into phenylpropanoid-derived products. The latter, in turn, depending on the plant species, can account for greater than 50% of the plant dry weight, e.g. woody tissue. Many of these plant products represent highly valuable substitutes for existing biofuel/polymer precursors; however, our understanding of how this carbon allocation is both controlled and can be optimized within the plastid and cytosol remains at a very preliminary level. Specifically, in our recent studies, we have discovered and characterized the previously unknown enzyme in plants, arogenate dehydratase (ADT), that generates phenylalanine in plastids. ADT, part of a multigene family in Arabidopsis thaliana (six isoforms) and Populus trichocarpa (five isoforms), is considered to be differentially involved in the biosynthesis/ carbon allocation to phenylpropanoid metabolites and protein formation, respectively. Using a range of molecular biological techniques, we have produced or are producing the plant lines harboring all of the permutations of individual gene suppressions and over-expressions in the ADT family. This provides a remarkable opportunity to systematically identify significant gene products that control carbon allocation to the potential biofuels/bioproducts, which could prove highly useful in meeting the DOE directive of reducing our dependence on foreign oil, as well as supplanting oil-derived fuels and their valuable side-products with ones obtained from plants. This proposed research requires the use of the Free Flow Electrophoresis organelle separation system located at the EMSL. This system will allow us to obtain very pure chloroplast/plastid isolations, which will provide higher confidence in the localization of identified proteins, associated with them. Secondly, in-house ultra-high pressure liquid chromatography systems (not available anywhere else) will enable us to obtain superior peptide separations of the extremely complex proteome of Arabidopsis and poplar tree samples, which will allow for a larger number of peptides to be identified ultimately in the high resolution Orbitrap mass spectrometers. Taken altogether, this proteome platform, will provide a comprehensive proteome profile of chloroplasts with a depth of coverage and insights into plant biosynthesis/carbon allocation not attainable anywhere else but at the EMSL.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-10-13
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator