Plant peroxisomes are essential components in various physiological and metabolic pathways, yet our knowledge of the protein composition and metabolic and regulatory networks associated with these organelles is far from complete. This 2010 project will generate a 'parts list' of peroxisomal matrix and membrane, using bioinformatics and proteomics, followed by fluorescence microscopy and protein fractionation. Further, the functions of peroxisome-targeted proteins will be analyzed with various assays on sequence-indexed T-DNA insertion mutants. Finally, peroxisomal protein complexes and networks will be explored, using gel-based methods in combination with mass spectrometry. Approximately 300-500 genes encoding peroxisomal proteins will be associated with biological functions, which is a critical step toward developing a comprehensive model for plant peroxisome function. This project will provide the scientific community with a much-needed comprehensive inventory of peroxisomal proteins and a large set of tagged reporter constructs for follow-up studies. All information will be integrated into the AraPeroX database (www.araperox.uni-goettingen.de/) and mirrored on the project website www.peroxisome.msu.edu. Information and materials generated will be available to the public in a timely fashion through our project website, TAIR, and ABRC. Given that different cell compartments are often linked via peroxisomal functions and that peroxisomes are crucial during stress responses, this project will benefit several fields in the plant community and achieve synergistic impact with other ongoing 2010 projects, such as the plastid functional genomics project. Results from this research will also expand our knowledge of eukaryotic cell biology and provide useful information to engineering crop plants for increased stress resistance and improved oil production.
Broader Impacts: Research and education will be integrated by multidisciplinary training of undergraduates, high school students and teachers, and teachers from primarily undergraduate colleges. The data, resources, and techniques generated in the project will be useful to a broad community of scientists.
Peroxisomes are eukaryotic organelles surrounded by a single membrane and executing a wide range of metabolic functions. The importance of peroxisomes is manifested by the lethal genetic diorders in human and plant lethality when peroxisome biogenesis or major functions are severely defective. Plant peroxisomes mediate biochemical and physiological processes such as fatty acid β-oxidation, the glyoxylate cycle, photorespiration, hydrogen peroxide metabolism, jasmonic acid biosynthesis, indole 3-butyric acid (IBA) metabolism, metabolism of polyamine, sulfite, and nitrogen. Plant peroxisomes serve as essential "nodes" in a number of metabolic networks within the plant cell through physical and metabolic links with other cellular compartments. The major protein constituents of plant peroxisomes had been well characterized. However, the full functional spectrum for these organelles was far from being completely decoded. The major goal for this 2010 project was to discover novel peroxisomal proteins and map peroxisomal functional networks. Using one-dimensional gel electrophoresis (1-DE) followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS), we performed in-depth proteome analysis of three Arabidopsis peroxisomal subtypes, i.e., those from green leaves, etiolated germinating seedlings, and senescent leaves, respectively. Use fluorescence microscopy, we tested the subcellular localization of over 100 putative novel peroxisomal proteins identified from proteomics and in silico PTS (peroxisome targeting signal) searches of the Arabidopsis genome, and confirmed the peroxisomal targeting of ~50 of them. Furthermore, we analyzed ~90 sequence-indexed T-DNA insertion mutants of over 50 novel peroxisomal genes through a series of physiological, biochemical, and cell biological assays to assess the roles of the corresponding proteins in peroxisomes. Analysis of the new peroxisomal proteins conclusively identified from this project revealed novel metabolic and regulatory functions of peroxisomes in processes such as detoxification, nucleic acid metabolism, protein degradation, and plant defense. Peroxisomal metabolic pathways in different developmental stages were also compared. Detailed analysis of a few plant-specific peroxisomal proteins has been conducted. We have deposited our subcellular localization results and constructs to the Arabidopsis database and Arabidopsis Biological Resource Center (ABRC) and listed identified genes at our project website. A comprehensive peroxisome database that integrates proteomics, subcellular localization, and reverse genetic data with hypotheses of gene function is being constructed. Results from this project have taken the plant peroxisome research field an important step toward defining the complete plant peroxisomal proteome and functional networks. They also provide valuable information to peroxisomal research in monocot plants, many of which are important cereal crops with economical significance. We have provided research training for seven postdoctoral level researchers, six graduate students, 25 undergraduates, four technicians/visiting scholars, and three high school students. Eleven of the domestic trainees were from under-represented groups. This project also fostered collaborations with other research groups and training programs in the US, Europe and Asia. This project funded or partially funded works that resulted in close to 20 publications in the form of research articles or reviews. Through various venues, we have presented over 30 research talks and more than 20 posters on the work related to this 2010 project.
