EAGER: spRNA-seq: high-throughput transcriptome analysis of single plastids

质体是植物细胞中普遍存在的重要细胞器。它们有多种类型，叶绿体是最著名的质体类型。通过光合作用，叶绿体收集太阳能并将二氧化碳转化为植物生物量，从而维持地球上几乎所有的生命。事实上，质体的功能并不局限于光合作用，其他类型的质体在植物的发育、代谢、信号传导和免疫等方面发挥着多种重要作用。质体及其多种功能可以被用来造福人类和环境。然而，我们对质体的了解受到当前技术的严重限制。每个植物细胞都含有数十到数百个质体，而目前的方法既可以一次研究几个质体，比如通过显微镜，也可以大量研究数百万个质体，比如通过分子技术。前者缺乏效率，而后者平均可能不同的质体类型。本实验旨在建立一种研究质体的新技术，即单质体RNA测序（spRNA-seq）。这项技术在一次实验中确定了数十万个个体质体的分子特征，使有效和充分了解质体多样性及其功能成为可能。这项技术有望彻底改变质体的研究，并将对质体如何影响植物生物学、生态学和进化产生新的见解。这些知识可以用来利用质体的力量来改善地球上的生活。本合作项目的pi计划使用拟南芥和油菜的质体作为技术对照和生物学比较模型，建立spRNA-seq方法。该项目包括三个组成部分。首先，设计、优化和实施基因组学策略，以实现可重复、成本效益高和用户友好的spRNA-seq。特别是，质体将被分离，并采用分裂-池策略，在文库制备过程中实现质体内组合条形码，每个质体通过独特的条形码组合进行生物信息识别。接下来，将应用一套计算和统计方法来评估spRNA-seq的技术结果，更重要的是，通过转录组异质性来收集对新质体类型的见解。由于质体转录组在很大程度上受到剪接、编辑、核内溶和核外溶等转录后机制的影响，因此这些转录后事件的潜在质体间异质性将受到质疑。最后，单分子RNA原位杂交将用于验证质体异质性。这种互补的方法增加了空间分辨率，以进一步了解细胞或组织内的质体异质性。此外，该项目将培养下一代科学家，包括加州大学河滨分校未被充分代表的少数族裔学生，培养他们的批判性思维、实验设计和执行以及科学交流能力。该研究小组将通过出版物、会议演讲和培训视频传播这一授权技术，以推动科学发现。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。

Plastids are ubiquitous and essential organelles in plant cells. They come in diverse types, with chloroplasts being the most well-known plastid type. Through photosynthesis, chloroplasts harvest solar energy and convert carbon dioxide into plant biomass, and thus sustain almost all life on Earth. In fact, the functions of plastids are not limited to photosynthesis – other types of plastids exist and perform a variety of essential functions in development, metabolism, signaling, and immunity in plants. Plastids and their diverse functions can be harnessed for the benefit of humans and the environment. However, our knowledge of plastids is severely limited by current technologies. Each plant cell harbors tens to hundreds of plastids and current approaches can either study a few plastids at a time, such as by microscopy, or millions of them in bulk, such as by molecular techniques. The former lacks efficiency while the latter averages potentially distinct plastid types. The proposed experiments are aimed at establishing a new technology to study plastids, namely single-plastid RNA sequencing (spRNA-seq). This technology determines the molecular signatures of hundreds of thousands of individual plastids in one experiment, making it possible to efficiently and fully understand plastid diversity and how they function. The technology is expected to revolutionize research on plastids and will generate novel insights into how plastids affect plant biology, ecology, and evolution. This knowledge can be used to harness the power of plastids for the betterment of life on Earth.The PIs of this collaborative project plan to use plastids from Arabidopsis thaliana and Pisum sativum, as both technical controls as well as biologically comparative models, to establish the spRNA-seq method. The project comprises three integrative components. First, a genomics strategy is designed, optimized, and implemented to achieve spRNA-seq that is reproducible, cost-effective, and user-friendly. In particular, plastids will be isolated and a split-and-pool strategy will be employed to enable in-plastid combinatorial barcoding during library preparation, with each plastid being identified bioinformatically through the unique combination of barcodes. Next, a suite of computational and statistical approaches will be applied to evaluate the technical outcomes of spRNA-seq, and more importantly, to glean insights into new plastid types through transcriptomic heterogeneity. As the plastid transcriptome is heavily molded by posttranscriptional mechanisms such as splicing, editing, and endonucleolytic and exonucleolytic processing, potential inter-plastid heterogeneity with respect to these posttranscriptional events will be interrogated. Finally, single-molecule RNA in situ hybridization will be deployed for validation of plastid heterogeneity. This complementary approach adds spatial resolution to provide further insights into plastid heterogeneity within a cell or tissue. Further, the project will train the next generation of scientists, including underrepresented minority students at University of California Riverside, in critical thinking, experimental design and execution, and scientific communications. The research team will disseminate this empowering technology through publications, conference presentations, and training videos to propel scientific discovery.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.