Regulatory Hierarchies and Roles of Non-Coding RNAs in Maize Anthers

PI：Blake C. Meyers（特拉华州大学）CoPI：Virginia Walbot（斯坦福大学）和Greg Abrams（德克萨斯州高级计算中心/德克萨斯大学奥斯汀分校）该项目产生的信息将为玉米和相关禾本科作物花药（雄性生殖器官）发育事件的调控提供新的见解。 新的见解和具有广泛用途的资源最终应有助于控制农业中的植物肥力，特别是谷类作物。该项目建立并改进了PI的各种广泛的外联和教育组成部分，并通过科学领导力继续对当地和科学界以及下一代科学家产生强大的积极影响。“虚拟花药”的实施将使项目数据广泛可用，同时帮助学生和公众了解花药是如何发育的。一个以本科生为重点的项目，测序和注释基础草基因组将为该项目提供宝贵的数据，并为所有在开花植物中使用基因组学分析的人提供宝贵的数据，同时培养下一代植物生物学家。所有项目成果都将通过项目网站和GEO和MaizeGDB等长期储存库向研究界提供。所有生物资源，如抗体，克隆和载体将根据要求提供。种子将通过玉米遗传学合作社长期存放和分发。“虚拟花药”软件将开放源码，并可通过得克萨斯州高级计算中心网站（https：//www.tacc.utexas.edu/tacc-software/）获得。雄性不育是自然物种中常见的表型，作为促进异交的一种机制。此外，植物育种者对花粉生产的控制是一项基本技术，继续支持许多作物的杂交种子生产。在农业方面，人们也越来越担心气候破坏：热和冷都会导致作物雄性不育。这项研究旨在更广泛地了解花药发育和潜在的细胞过程，包括染色质重塑的协调，植物细胞从有丝分裂到减数分裂。 具体目标是：（1）组织单个花粉囊细胞类型中的基因表达层次;（2）阐明小RNA的功能，特别是两类不寻常的“定相”二级小RNA（phasiRNA），它们在草花粉囊中高度富集。这些phasiRNA从大量协调表达的长非编码RNA前体加工成21或24个核苷酸的次级小RNA。利用精确分期的玉米花药和激光显微切割的细胞类型，该项目将产生大量的转录组学和蛋白质组学数据，组织转录因子的转录层次，在时空背景下产生大量关于小RNA的数据，并分析phasiRNA的功能和生物起源。使用现有和工程突变的遗传分析将用于测试关键转录和phasiRNA生物发生因子在花药发育中的重要作用。遗传学和“组学”实验将测试关于phasiRNA在花药细胞和减数分裂中功能的新假设。大部分分析将在玉米中进行，因为玉米具有精细的发育规律，花药解剖的大尺寸和容易性，以及关于玉米花药的现有分子和遗传知识。关键的见解将在水稻中进行评估，以区分一般和物种特异性点，并与其他草进行额外的比较。为了产生新的假设，所有数据将被整合到一个“虚拟花药”可视化系统。

PI: Blake C. Meyers (University of Delaware)CoPIs: Virginia Walbot (Stanford University) and Greg Abrams (Texas Advanced Computing Center/University of Texas - Austin) Information generated from this project will provide new insights into the regulation of developmental events in the anther, the male reproductive organ, of maize and related grasses - crops that feed much of the world. New insights and resources of broad utility should ultimately contribute to controlling plant fertility in agriculture, particularly in the cereal crops. The project builds and improves on the varied and extensive outreach and education components of the PIs and their contributions via scientific leadership to continue a strong, positive impact on local and scientific communities, and on the next generation of scientists. Implementation of the "Virtual Anther" will make the project data widely accessible while helping students and the public understand how anthers develop. An undergraduate-focused project to sequence and annotate a basal grass genome will provide invaluable data for the project and to all those employing phylogenomics analysis in flowering plants, while training the next generation of plant biologists. All project outcomes will be accessible to the research community through a project website and long-term repositories such as GEO and MaizeGDB. All biological resources such as antibodies, clones and vectors will be available upon request. Seed will be deposited and distributed long-term through the Maize Genetics Cooperative. Software for the "Virtual Anther" will be made open-source and available via the Texas Advanced Computing Center website (https://www.tacc.utexas.edu/tacc-software/).Male sterility is a common phenotype in natural species, functioning as one mechanism to promote outcrossing. Furthermore, control of pollen production by plant breeders is a fundamental technology that continues to underpin hybrid seed production in many crops. In the agricultural context, there is also a growing concern over climate disruption: both heat and cold cause male sterility in crop plants. This study seeks to more broadly understand the coordination of anther development and underlying cellular processes, including chromatin remodeling, as plant cells switch from mitosis to meiosis. The specific goals are to: (1) organize hierarchies of gene expression in individual anther cell types and (2) elucidate functions of small RNAs, particularly two unusual classes of "phased" secondary small RNAs (phasiRNAs) that are highly enriched in grass anthers. These phasiRNAs are processed from an extensive set of coordinately-expressed long non-coding RNA precursors into secondary small RNAs of 21 or 24 nucleotides. Using precisely staged maize anthers and laser microdissected cell types, the project will generate copious transcriptomic and proteomic data, organize transcriptional hierarchies of transcription factors, generate extensive data on small RNAs in a spatiotemporal context, and analyze the function and biogenesis of phasiRNAs. Genetic analysis using existing and engineered mutations will be used to test for essential roles of key transcription and phasiRNA biogenesis factors in anther development. Genetic and "omics" experiments will test new hypotheses about phasiRNA functions in anther cells and in meiosis. Most of the analysis will be conducted in maize because of its exquisite developmental regularity, the large size and ease of anther dissection, and existing molecular and genetic knowledge about maize anthers. Key insights will be evaluated in rice to distinguish general and species-specific points, with additional comparisons to other grasses. To generate new hypotheses, all data will be integrated into a "Virtual Anther" visualization system.