RESEARCH-PGR: Role of cytokinin in regulating shoot apical meristem function in rice

植物的叶子和花来源于被称为分生组织的干细胞组织。分生组织发育和活动的调控是理解植物生长发育的核心，并影响许多重要的农学性状，如植物结构、生物量和粮食产量。该项目的目标是确定植物激素细胞分裂素如何调节水稻的分生组织功能，水稻是一种重要的作物，也是了解其他谷物生长发育的模型系统。为此目的，将采用最近开发的单细胞测序方法。在芽分生组织和生殖花序分生组织的单个细胞中的基因表达将被检查，以揭示调节叶，花和籽粒发育的细胞机制。此外，基因组的包装将被表征，以识别可能在控制基因在特定细胞中表达的时间、地点和程度方面发挥作用的DNA元素。破坏细胞分裂素功能所带来的分生组织发育的改变将揭示这种激素如何在细胞水平上协调水稻的正常发育。这些研究将阐明营养和花序结构发育的机制，重点关注细胞分裂素在这些过程中的作用。在更广泛的背景下，这项工作有可能提高水稻和其他重要农业谷物品种的产量。拟议的研究将通过提供学生培训和制定旨在促进科学教育的计划来加强研究和教育的基础设施。植物激素细胞分裂素（cytokinin）在水稻营养芽顶端分生组织（SAM）和生殖花序分生组织（IM）的活性调控中起关键作用。在这里，细胞分裂素在调节水稻SAM和IM中的作用将在单细胞和单核水平上使用遗传、分子和单细胞“组学”方法的综合阵列进行研究。首先，将使用单细胞转录组学和单核染色质谱分析来表征单个SAM/IM细胞的转录丰度和染色质可及性。这将提供对SAM和IM的监管电路的基本理解。SAM/IM亚域内的标记基因和开放染色质区域将与水稻穗性状的GWAS面板中鉴定的多态性相关，从而可能确定穗结构的重要基因组调控区域。其次，使用细胞分裂素受体突变体对细胞分裂素信号传导进行遗传干扰，以揭示这种调节网络如何影响细胞类型、它们的分布和发育轨迹，以及细胞簇内基因和调节元件的活性。第三，由于b型应答调节因子（type-B Response Regulator， RR）转录因子控制细胞分裂素的初始转录输出，因此b型RR的直接靶点将通过ChIP-seq来确定。整合这些数据集将确定特定细胞和SAM/IM类型中细胞分裂素信号传导的关键靶点，然后通过遗传分析检查其中的一个子集。总的来说，拟议的研究将产生互补的基因组水平数据集，这些数据集将确定细胞分裂素调节分生组织活性的关键介质，潜在地揭示分生组织调节的新特性。该奖项是由综合生物系统部门的植物基因组研究计划和分子与细胞生物科学部的遗传机制集群共同资助的。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Plant leaves and flowers are derived from stem cell tissues called meristems. The regulation of meristem development and activity is central to understanding plant growth and development, and impacts many agronomically important traits such as plant architecture, biomass, and grain yield. The goal of this project is to determine how the plant hormone cytokinin regulates meristem function in rice, an important crop and a model system for understanding growth and development of other cereals. For this purpose, the recently developed methodologies for single cell sequencing will be employed. Gene expression in individual cells of the shoot meristem and of the reproductive inflorescence meristem will be examined to uncover cellular mechanisms that regulate the development of leaves, flowers, and grain. In addition, the packaging of the genome will be characterized to identify DNA elements that likely play a role in controlling when, where, and to what degree the genes are expressed in specific cells. Alternations in meristem development, brought about by disrupting cytokinin function will reveal how this hormone orchestrates proper rice development at the cellular level. These studies will illuminate the mechanisms underlying the development of vegetative and inflorescence architectures, with an important emphasis on cytokinin’s role in these processes. In broader context, this work has the potential to realize increased yields in rice and other agriculturally important cereal species. The proposed research will enhance the infrastructure of research and education by providing student training and the development of programs aimed at fostering science education.The phytohormone cytokinin plays key roles in regulating the activities of the vegetative shoot apical meristem (SAM) and reproductive inflorescence meristem (IM) in rice. Here, the role of cytokinin in regulating the rice SAM and IM will be examined at the single cell and single nucleus levels using an integrative array of genetic, molecular, and single-cell ‘-omic’ approaches. First, transcript abundance and chromatin accessibility in individual SAM/IM cells will be characterized using single-cell transcriptomics coupled with single-nucleus chromatin profiling. This will provide foundational understanding of the regulatory circuits in the SAM and IM. Marker genes and regions of open chromatin within subdomains of the SAM/IM will be correlated to polymorphisms identified in GWAS panels for panicle traits in rice, potentially identifying important genomic regulatory regions for panicle architecture. Second, cytokinin signaling will be genetically perturbed using cytokinin receptor mutants to reveal how this regulatory network influences cell types, their distribution and developmental trajectories, and the activity of genes and regulatory elements within the cell clusters. Third, because type-B Response Regulator (RR) transcription factors control the initial transcriptional output from cytokinin, the direct targets of type-B RRs will be identified by ChIP-seq. Integrating these datasets will identify key targets of cytokinin signaling in specific cells and types of the SAM/IM, a subset of which will then be examined by genetic analysis. Collectively, the proposed studies will result in complementary genome-level datasets that will identify key mediators of cytokinin in regulating meristem activity, potentially revealing emergent properties of meristem regulation.This award was co-funded by the Plant Genome Research Program in the Division of Integrative Organismal Systems and the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences.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.