The Role of Meiotic-Stage Non-Coding RNA in the Modulation of Anther & Pollen Development in Grasses

本项目将研究一类支持草花花粉发育的小RNA的生命周期和功能，特别是在花药中。这些实验的重点是这些24核苷酸RNA的生物发生、精确定位和定量，利用它们实现花药功能的蛋白质，以及它们在植物育性中的作用。先前的工作表明，即使在轻微的温度压力下，这些小RNA也是男性生育能力强健所必需的：如果没有它们，花药发育就会失败。该项目将把重点放在玉米花药上，因为很容易准确确定它们处于什么发育阶段，它们很容易被解剖，有许多发育突变会影响已经分离出的基因的花药，而且因为了解雄性育性对杂交玉米种子的生产很重要。更详细地了解花粉发育的分子基础能够改进种子生产，包括杂交种子；在牧草方面，杂交玉米和水稻显著提高了世界粮食产量。该项目的成果可能包括发现新的遗传途径，以更精确地控制植物的雄性育性，并使植物的育性对环境变化更具弹性。该项目将利用植物基因组学和定向突变、尖端成像技术、生物信息学，重点是发育生物学。该项目的更广泛影响包括通过与长期且成功的本科和高中实习计划的持续整合，对学生进行植物和计算生物学方面的培训。该项目将专门专注于24-NT阶段性次级siRNAs，即“24-nt phasiRNAs”，它在减数分裂阶段的花药中高度丰富。这项工作将以玉米为主要物种进行分析，因为存在着重要的、现有的遗传和分子资源。该项目将解决几个重要问题。减数分裂细胞和体细胞在小RNA的积累和它们的功能上有区别吗？哪些转录因子指导了决定细胞命运和包括phasiRNAs在内的特定细胞类型属性的分化所需的转变？如何利用减数分裂阶段的小RNA来引导花药细胞的命运和分化？MicroRNA的触发物、前体mRNAs和生物发生或效应蛋白何时何地积累？该项目将对影响phasiRNAs的功能缺失敲除突变的表型和分子遗传分析进行表征，包括Dice-like 5(Dcl5)基因的突变，初步数据表明该基因存在男性不育。项目成员将通过他们的个人和小组项目在分析小型RNA的本地化、生物发生和生物化学方面接受广泛的植物小RNA生物学和基因组学、细胞生物学和显微镜以及计算方法方面的培训。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project will investigate the lifecycle and functions of a class of small RNAs that support pollen development in grass flowers, specifically in anthers. The experiments focus on the biogenesis, precise localization, and quantification of these 24-nucleotide RNAs, the proteins that utilize them for anther functions, and their role in plant fertility. Prior work demonstrated that these small RNAs are required for robust male fertility under even slight temperature stress: without them anther development fails. The project will focus on maize anthers because it is easy to determine accurately what developmental stage they are in, they are easy to dissect, there are numerous developmental mutants that affect anthers for which the gene has been isolated, and because understanding male fertility is important to the production of hybrid corn seed. A more detailed understanding of the molecular basis of pollen development enables improvements in seed production, including hybrid seeds; in the grasses, hybrid corn and rice have significantly boosted world food production. Outcomes of this project could include discovering new genetic pathways for more precise control of male fertility in plants, and plants with fertility that is more resilient to environmental change. The project will utilize plant genomics and targeted mutagenesis, cutting-edge imaging techniques, bioinformatics, with a focus on developmental biology. Broader impacts of the project include training of students in plant and computational biology via continued integration with long-running and successful undergraduate and high school internship programs. The project will focus specifically on the class of 24-nt phased, secondary siRNAs, known as "24-nt phasiRNAs" that are highly enriched in meiotic-stage anthers. The work will be done in maize as the primary species for analysis because there are significant, existing genetic and molecular resources. The project will address several important questions. Do meiotic cells differ from somatic cells in the accumulation of small RNAs and their functions? Which transcription factors guide the transitions required for setting cell fate and differentiation of cell type-specific properties that include phasiRNAs? How are meiotic-stage small RNAs deployed to guide anther cell fate and differentiation? Where and when do the microRNA triggers, precursor mRNAs, and biogenesis or effector proteins accumulate? The project will characterize phenotypes and molecular genetic analysis of loss-of-function knock-out mutations that impact phasiRNAs, including mutations of the Dicer-like5 (Dcl5) gene, for which preliminary data demonstrate male sterility. Project members will be trained broadly in plant small RNA biology and genomics, cell biology and microscopy, and computational methods through their individual and group projects in the analysis of the localization, biogenesis, and biochemistry of small RNAs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.