Macromolecular Networks Underlying Chloroplast Biogenesis

PI: Alice Barkan (University of Oregon - Eugene)CoPI: Klaas van Wijk （Cornell University）叶绿体是植物的一个重要特征，具有多种代谢功能，深刻影响植物的产量和对环境胁迫的反应。光合作用是叶绿体存在的主要原因，涉及复杂的机制，其生物发生需要一系列功能和物理上相关的基因表达和组装过程。该项目的目标是促进对叶绿体光合机构生物发生基础上的DNA/RNA/蛋白质相互作用网络的理解。选择用于研究的大分子组装预计将富含未知功能的蛋白质，并将通过协调一致的遗传，蛋白质组学和核糖组学分析进行解剖。玉米将成为主要的实验生物，因为它的特性使它特别适合这些方法——它是一种重要的作物物种，也是理解C4光合作用的主要模型。与拟南芥的选择性比较将有助于将结果外推到单子叶和双子叶种。叶绿体生物发生所需的核基因将通过结合下一代测序技术和深度收集非光合玉米突变体的高通量正向遗传策略来发现。现有的表型数据将突变置于未被研究的功能类别中，将用于选择100个品系进行分析。免疫纯化的大分子组件的蛋白质成分将通过高灵敏度质谱法鉴定。抗体靶点将包括：与翻译多体到用于合成和组装叶绿体编码蛋白质的免疫纯化“工厂”相关的新生肽链；与叶绿体染色体相关的蛋白质；以及叶绿体基因表达和蛋白质折叠/组装机制界面上的蛋白质。新鉴定的蛋白质将通过反向遗传学和表型分析以及核酸配体的鉴定（如相关）进行功能表征。最后，全基因组RNA/DNA共免疫沉淀法将用于鉴定与通过遗传和蛋白质组学方法鉴定的核酸相互作用蛋白相关的核酸，并探索细胞器专用RNA结合蛋白家族的功能。本研究将与各级学生的教育相结合。俄勒冈大学将开展一项针对高中生的暑期研究项目，该项目以实验为中心，与该项目的目标相结合，并为担任助教的本科生和研究生提供指导培训。该项目将参与康奈尔大学/BTI现有的项目，为来自小型机构的本科生提供研究培训，并参与高中教师的课程开发研讨会。将产生的社区资源包括：(i)相互作用数据、蛋白质鉴定和遗传数据，所有这些数据将提交给公共数据库存储库，并用于开发数百个玉米基因的功能注释；（ii）约100个突变玉米系，具有已鉴定的突变和已鉴定的突变表型，以及数千个序列索引转座子插入和相关玉米种群；(3)玉米细胞器微阵列和针对各种叶绿体蛋白的抗体。相互作用数据将有助于社区努力开发高质量的植物相互作用数据集。相互作用数据和基因注释将通过集中的资源传播，包括Gramene （http://www.gramene.org/）、MaizeGDB （http://www.maizegdb.org/）、PlantGDB （http://www.plantgdb.org/）和植物蛋白质组数据库（PPDB; http://ppdb.tc.cornell.edu/）。玉米库存将提供给玉米遗传鸡舍库存中心进行分配。微阵列和抗体将根据要求按成本价提供。

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.