Macromolecular Networks Underlying Chloroplast Biogenesis

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

PI: Alice Barkan (University of Oregon - Eugene) CoPI: Klaas van Wijk (Cornell University) Chloroplasts are a defining feature of plants, with diverse metabolic functions that profoundly impact plant yield and response to environmental stress. Photosynthesis is the primary raison d'etre of the chloroplast, and involves complex machinery whose biogenesis requires a series of functionally and physically-connected gene expression and assembly processes. The goal of this project is to advance understanding of the network of DNA/RNA/protein interactions that underlie the biogenesis of the photosynthetic apparatus in chloroplasts. Macromolecular assemblies selected for study are anticipated to be rich in proteins of unknown function, and will be dissected through concerted genetic, proteomic, and ribonomic analyses. Maize will be the primary experimental organism because its attributes make it especially well suited for these methods - it is an important crop species and a prime model for understanding C4 photosynthesis. Selective comparisons with Arabidopsis will facilitate extrapolation of results to both monocot and dicot species. Nuclear genes required for chloroplast biogenesis will be discovered through a high-throughput forward-genetic strategy that combines next generation sequencing technologies with a deep collection of non-photosynthetic maize mutants. Existing phenotypic data that place mutants into understudied functional classes will be used to select 100 lines for this analysis. Protein components of immunopurified macromolecular assemblies will be identified through high sensitivity mass spectrometry. Antibody targets will include: nascent peptide chains associated with translating polysomes to immunopurified "factories" for the synthesis and assembly of chloroplast-encoded proteins; proteins associated with the chloroplast chromosome; and proteins at the interface of the chloroplast gene expression and protein folding/assembly machineries. Newly-identified proteins will be functionally characterized by reverse-genetics and phenotypic analyses, and by identification of their nucleic acid ligands (where relevant). Finally, genome-wide RNA/DNA co-immunoprecipitation assays will be used to identify the nucleic acids associated with nucleic-acid interacting proteins identified via the genetic and proteome approaches, and to explore the functions of organelle-dedicated RNA binding protein families. This research will be integrated with the education of students at all levels. A summer research program for high school students that centers on experiments integral to the goals of this project, and that provides mentorship training for undergraduate and graduate students who serve as teaching assistants will be developed at the University of Oregon. The project will participate in existing programs at Cornell/BTI to provide research training for undergraduate students from small institutions and to participate in curriculum development workshops for high school teachers. Community resources to be generated include: (i) interaction data, protein identifications, and genetic data, all of which will be submitted to public database repositories and used to develop functional annotations for several hundred maize genes; (ii) ~100 mutant maize lines with an identified mutation and characterized mutant phenotype, as well as several thousand sequence-indexed transposon-insertions and associated maize stocks; and, (iii) maize organellar microarrays and antibodies to various chloroplast proteins. The interaction data will contribute to community efforts to develop a high quality plant interactome data set. Interaction data and gene annotations will be disseminated through centralized resources that include Gramene (http://www.gramene.org/), MaizeGDB (http://www.maizegdb.org/), PlantGDB (http://www.plantgdb.org/), and the Plant Proteome Database (PPDB; http://ppdb.tc.cornell.edu/). Maize stocks will be provided to the Maize Genetics Coop Stock Center for distribution. Microarrays and antibodies will be made available at cost by request.