EAGER: Comparative single cell transcriptomics and regulomics: A proof-of-concept application of cutting-edge -omics techniques with non-model systems

植物产生各种化学物质，称为天然产物，其功能是威慑害虫，吸引传粉者，并与微生物相互作用。虽然最近的技术使人们能够发现负责天然产物生产的酶，但人们对植物如何调节这些化合物的生产知之甚少。新出现的研究表明，越来越多的植物天然产物是在不同的细胞类型中产生的，这表明它们的生物合成受到严格的调控。该项目将侧重于单萜吲哚生物碱，这是一类不同的专门代谢物，其中一些具有强大的生物活性和药学活性。目标是发现长春花、马达加斯加长春花及其近亲喜树的单萜吲哚生物碱生物合成的调节成分。通过这个项目，将开发数据和方法来阐明基因调控的分子机制。本科生将接受与植物生物学相关的最先进的遗传方法、计算分析和数据分析方法的培训。通过这一项目，将创造新的生物技术和植物天然产物工程生产的工具。越来越多的证据表明，植物的专门化代谢在器官、组织和细胞类型水平上受到精细和严格的调节，从而导致天然产物的不同细胞类型和亚细胞定位。负责天然产物生物合成的基因是如何被调控的，目前还不是很清楚。单萜烯吲哚生物碱(MIA)是龙胆目植物产生的一类特殊代谢产物，包括长春花属植物和尖叶龙胆属植物。在玫瑰花中，MIA的生物合成途径被顺序地划分为三种不同的细胞类型，这表明控制中间体的生物合成和运输的复杂调控网络。然而，在其他物种中，MIA的定位和细胞类型特异性仍然是一个谜。虽然MIA生物合成途径的早期步骤在玫瑰花和尖顶锦鸡儿之间是保守的，但下游阶段是不同的。在基因、细胞类型和调控水平上了解两条相关的MIA生物合成途径将有助于揭示这些复杂天然产物的调控和进化，以及如何在异源系统中重建和修饰该途径。这个项目将展示单细胞转录和染色质可及性图谱的可行性，以了解MIA生物合成基因在叶细胞中的细胞类型特异性，以及C.roseus和C.acumata之间的保守程度。该项目还将证明DNA亲和测序(DAP-seq)在玫瑰花冠和尖顶锦鸡儿中作为一种连接通过计算方法确定的转录因子和顺式调控元件的方法的可行性。综上所述，该项目将为两种用于植物的尖端基因组学技术建立最佳实践，并有可能加深我们对植物专门代谢的监管环境的理解。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。

Plants produce diverse chemicals, known as natural products, that function to deter pests, attract pollinators, and interact with microbes. While recent technologies have enabled the discovery of the enzymes responsible for natural product production, there is limited knowledge of how plants regulate production of these compounds. Emerging studies have shown that a growing number of plant natural products are produced in distinct cell types, suggesting strict regulation of their biosynthesis. This project will focus on the monoterpene indole alkaloids, a class of diverse specialized metabolites, some of which have potent biological and pharmaceutical activities. The goal will be to discover the regulatory components of monoterpene indole alkaloid biosynthesis in Catharanthus roseus, Madagascar periwinkle, and its relative, Camptotheca acuminata. Through this project, data and methods will be developed to elucidate the molecular mechanisms by which genes are regulated. Undergraduates will be trained in state-of-the-art genetic methods, computational analyses, and data analysis methods associated with plant biology. Through this project, new tools for biotechnology and engineering the production of natural products in plants will be created.Accumulating evidence suggests plant specialized metabolism is under exquisite and strict regulation at the organ, tissue, and cell type levels, thereby leading to distinct cell type and subcellular localization of natural products. How the genes responsible for the biosynthesis of natural products are regulated is not well understood. Monoterpene indole alkaloids (MIA) are a class of diverse specialized metabolites produced by plants in the Order Gentianales, including C. roseus and the Asterid species C. acumuinata. In C. roseus, the MIA biosynthetic pathway is sequentially partitioned into three discrete cell types, suggestive of complex regulatory networks controlling the biosynthesis and transport of intermediates. In other species, however, the localization and cell type specificity of MIA remain an enigma. While the early steps of the MIA biosynthetic pathway are conserved among C. roseus and C. acuminata, the downstream stages are divergent. The knowledge of two related MIA biosynthetic pathways at the gene, cell type, and regulatory levels will shed light on the regulation and evolution of these complex natural products, as well as how the pathway can be reconstructed and modified in heterologous systems. This project will demonstrate the viability of single cell transcriptomics and chromatin accessibility profiling for C. acuminata to understand the cell type specificity of MIA biosynthetic genes across leaf cells and the extent of conservation between C. roseus and C. acuminata. This project will also demonstrate the viability of DNA affinity sequencing (DAP-seq) in C. roseus and C. acuminata as a method of bridging transcription factors and cis-regulatory elements identified by computational methods. Taken together, this project will establish best practices for two cutting edge genomics technologies for plants and potentially deepen our understanding of the regulatory landscapes of plant specialized metabolism.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.