Molecular Dissection of Autophagy in Maize and its Roles in Nutrient Recycling and Stress Protection

PI：Richard D. Vierstra（威斯康星州-麦迪逊大学）CoPI：Marisa S.奥特吉（威斯康星州-麦迪逊大学）植物采用复杂的机制来运输和回收细胞内成分，这些成分是生长和发育、管家和营养胁迫下生存所必需的。一种是将细胞质物质隔离在自噬小泡中，随后将这些小泡运送到液泡中进行分解或储存。先前的工作通过对拟南芥和玉米中一组基本的自噬相关（ATG）组分的分子，遗传和成像分析，在定义自噬机制方面取得了实质性进展。 与农业相关的是最近的研究结果，即自噬对玉米的氮利用效率至关重要，并发现了一种在玉米种子中起作用的新的自噬途径。 本项目的目标是：（i）在生物化学和遗传水平上进一步确定玉米ATG系统;（ii）通过将atg突变体与表型、细胞生物学、蛋白质组学、代谢组学和转录组学分析相结合，提供氮胁迫下自噬如何影响玉米生长、发育和产量的综合观点;（iii）通过自噬囊泡的蛋白质组学表征鉴定自噬降解的货物;以及（iv）定义玉米种子蛋白质积累期间支撑ATG非依赖性自噬的分子机制。 总的来说，这项研究将提供作物自噬及其在氮利用效率中的作用的第一个系统观点。 更广泛地说，这项研究还将产生重要的试剂、技术、突变体、转基因品系、种质和货物目录，这些将为理解重要作物玉米中的自噬提供急需的基础。 有了这些知识，就有可能重新设计作物，使其更有效地利用氮和其他营养素，更好地将这些营养素重新调动到新生长和储存的区域，表现出改善的叶片衰老和果实成熟特性，并提供更好的谷物产量。该项目旨在提供一个跨学科的工具，用于培训下一代植物科学家，使用最先进的遗传学，蛋白质组学，转录组学，代谢组学，和细胞生物学方法来研究作物生理学。参与者包括研究科学家，研究生和几个本科生在威斯康星州或参加UW-夏季REU经验主办的综合生物科学夏季研究计划，以及高中学生赞助的威斯康星州青年学徒计划（雅普）在生物技术。 YAP生物技术旨在通过正式的技术课程与现实生活中的研究经验相结合，为高中三年级和四年级学生提供实践培训，以扩大生物科学的劳动力。额外的教育影响包括由CoPI开发的细胞生物学研讨会，CoPI是NSF赞助的植物成像中心的主管，与将在该项目期间完善的下一代断层成像技术有关。其中一些改进将通过由UW工程学院组织的工程设计课程项目介绍来开发，该项目将创建用于成像玉米胚乳的优化设备。 通过将数据存放在MaizeGDB（http：//www.maizegdb.org/）和NCBI基因表达综合库（GEO; http：//www.ncbi.nlm.nih.gov/geo/）公共存储库，将促进公众免费和不受阻碍地访问本项目生成的RNA-seq、代谢组学和蛋白质组学数据集。 突变种质和稳定的转基因品系将通过玉米遗传合作储备中心（http：//www.example.com）提供。maizecoop.cropsci.uiuc.edu

PI: Richard D. Vierstra (University of Wisconsin - Madison)CoPI: Marisa S. Otegui (University of Wisconsin - Madison)Plants employ sophisticated mechanisms to traffic and recycle intracellular constituents needed for growth and development, housekeeping, and survival under nutrient stress. One involves the sequestration of cytoplasmic material in autophagic vesicles and subsequent delivery of these vesicles to the vacuole for breakdown or storage. Prior work made substantial progress toward defining the mechanisms underpinning autophagy through molecular, genetic, and imaging analyses of a set of essential autophagy-related (ATG) components in both Arabidopsis and maize. Of relevance to agriculture were recent findings that autophagy is critical to nitrogen-use efficiency in maize and the discovery of a novel autophagic route that operates in maize seeds. The objectives of this project are to: (i) further define the maize ATG system at the biochemical and genetic levels; (ii) provide an integrated view of how autophagy impacts maize growth, development, and yield during nitrogen stress by combining atg mutants with phenotypic, cell biological, proteomic, metabolomic, and transcriptomic analyses; (iii) identify the cargo degraded by autophagy via proteomic characterizations of autophagic vesicles; and (iv) define the molecular mechanism underpinning ATG-independent autophagy during maize seed protein accumulation. Collectively, this research will provide the first systems view of autophagy in a crop plant and its role in nitrogen-use efficiency. More broadly, this research will also generate important reagents, techniques, mutants, transgenic lines, germplasm, and cargo catalogs that will provide a much needed foundation to appreciate autophagy in the important crop, maize. With such knowledge, it should be possible to re-engineer crops that more efficiently use nitrogen and other nutrients, that better remobilize these nutrients to areas of new growth and storage, that display improved leaf senescence and fruit ripening characteristics, and that provide better grain yields.As a broader impact, this project is designed to provide an interdisciplinary vehicle for training the next generation of plant scientists using state-of-the-art genetic, proteomic, transcriptomic, metabolomic, and cell biological approaches to study crop physiology. Participants include research scientists, graduate students, and several undergraduates at Wisconsin or attending the UW-summer REU experience hosted by the Integrated Biological Sciences Summer Research Program, as well as high school students sponsored by the Wisconsin Youth Apprenticeship Program (YAP) in Biotechnology. YAP-Biotechnology is designed to expand the workforce in biological sciences by providing practical training to high school juniors and seniors through formal technology classes combined with real-life research experiences. Additional educational impacts involve cell biology workshops developed by the CoPI, who serves as the supervisor of the NSF-sponsored Plant Imaging Center, related to the next generation tomographic imaging techniques that will be refined during this project. Some of these refinements will be developed through an Introduction to Engineering Design course project organized by the UW-School of Engineering that will create devices optimized for imaging maize endosperms. Free and unencumbered public access to the RNA-seq, metabolomic, and proteomic datasets generated by this project will be facilitated by deposition of the data at MaizeGDB (http://www.maizegdb.org/) and the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) public repositories. Mutant germplasm and stable transgenic lines will be made available through the Maize Genetics Cooperative Stock Center (http://maizecoop.cropsci.uiuc.edu).