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

植物产生多种化学物质，被称为天然产物，其功能是阻止害虫，吸引传粉者，并与微生物相互作用。虽然最近的技术已经能够发现负责天然产物生产的酶，但对植物如何调节这些化合物的生产的了解有限。新的研究表明，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节。本项目将重点研究单萜吲哚生物碱，这是一类多样化的特化代谢物，其中一些具有强大的生物和药物活性。目的是发现花楸花、马达加斯加长春花及其近亲喜树单萜吲哚生物碱生物合成的调控成分。通过该项目，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。通过这个项目，将创造生物技术和工程在植物中生产天然产物的新工具。越来越多的证据表明，植物的特化代谢在器官、组织和细胞类型水平上受到精细而严格的调控，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还不是很清楚。单萜吲哚生物碱（MIA）是龙胆目植物产生的一类多样化的特化代谢物，包括玫瑰龙胆和菊科植物龙胆。在C. roseus中，MIA生物合成途径依次分为三种离散的细胞类型，表明控制中间体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C. roseus和C. acuminata中是保守的，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种相关的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，以及如何在异种系统中重建和修饰该途径。本项目将通过单细胞转录组学和染色质可及性分析来证明刺荆芥单细胞转录组学和染色质可及性分析的可行性，以了解MIA生物合成基因在叶片细胞中的细胞类型特异性以及刺荆芥和玫瑰芥之间的保护程度。该项目还将证明在C. roseus和C. acuminata中DNA亲和测序（DAP-seq）作为一种桥接转录因子和顺式调节元件的方法的可行性。总而言之，该项目将为两种尖端的植物基因组学技术建立最佳实践，并有可能加深我们对植物特化代谢调控景观的理解。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。

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.