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

该项目将研究一类支持花粉发育的小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个核苷酸的RNA的生物发生，精确定位和定量，利用它们进行花药功能的蛋白质，以及它们在植物育性中的作用。先前的工作表明，即使在轻微的温度胁迫下，这些小RNA也是强大的雄性育性所必需的：没有它们，花药发育就会失败。 该项目将重点放在玉米花药上，因为很容易准确确定它们所处的发育阶段，它们很容易解剖，有许多发育突变体影响基因已被分离的花药，并且因为了解雄性育性对杂交玉米种子的生产很重要。对花粉发育的分子基础有了更详细的了解，就可以改进种子生产，包括杂交种子;在禾本科植物中，杂交玉米和水稻大大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生育力，以及对环境变化更具适应力的植物。该项目将利用植物基因组学和靶向诱变，尖端成像技术，生物信息学，重点是发育生物学。该项目的更广泛的影响包括通过与长期运行和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。该项目将特别关注一类24-nt定相的二级siRNA，称为“24-nt phasiRNA”，它们在减数分裂期花药中高度富集。这项工作将在玉米作为主要物种进行分析，因为有重要的，现有的遗传和分子资源。该项目将解决几个重要问题。减数分裂细胞与体细胞在小分子RNA的积累及其功能上是否不同？哪些转录因子指导包括phasiRNA在内的细胞类型特异性特性的设定细胞命运和分化所需的转换？减数分裂阶段的小RNA如何引导花药细胞的命运和分化？microRNA触发物、前体mRNA、生物合成或效应蛋白在何处以及何时积累？该项目将表征影响phasiRNA的功能缺失敲除突变的表型和分子遗传学分析，包括Dicer样5（Dcl 5）基因的突变，初步数据显示雄性不育。项目成员将在植物小RNA生物学和基因组学、细胞生物学和显微镜学以及计算方法方面通过他们的个人和小组项目进行广泛的培训，这些项目分析了小RNA的定位、生物起源和生物化学。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值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.