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

叶绿体是植物细胞中普遍存在的重要细胞器。它们有多种类型，其中叶绿体是最广为人知的叶绿体类型。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质，从而维持地球上几乎所有的生命。事实上，叶绿体的功能并不局限于光合作用--植物中还存在其他类型的叶绿体，它们在发育、新陈代谢、信号传递和免疫等方面发挥着各种基本功能。可以利用叶绿体及其各种功能造福人类和环境。然而，我们对叶绿体的了解受到当前技术的严重限制。每个植物细胞含有数十到数百个叶绿体，目前的方法要么一次研究几个叶绿体，比如通过显微镜，要么大量研究它们，比如通过分子技术。前者缺乏效率，而后者平均可能存在不同的叶绿体类型。拟议的实验旨在建立一种研究叶绿体的新技术，即单叶绿体RNA测序(spRNA-seq)。这项技术在一次实验中确定了数十万个单独的叶绿体的分子特征，使人们有可能有效和充分地了解叶绿体的多样性及其功能。预计这项技术将彻底改变对叶绿体的研究，并将对叶绿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命。这个合作项目的PI计划使用拟南芥和豌豆的叶绿体作为技术控制和生物学比较模型，以建立spRNA-seq方法。该项目包括三个综合组成部分。首先，设计、优化和实施基因组学策略，以实现可重复性、成本效益和用户友好的spRNA-seq。特别是，在文库准备过程中，将分离叶绿体，并采用分裂和池策略，以实现叶绿体内组合条形码，通过条形码的独特组合，以生物信息识别每个叶绿体。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，通过转录异质性收集对新的叶绿体类型的洞察。由于叶绿体转录组在很大程度上受到转录后机制的影响，如剪接、编辑以及内切和外切加工，因此将询问与这些转录后事件有关的潜在的体间异质性。最后，将采用单分子RNA原位杂交来验证叶绿体的异质性。这种互补的方法增加了空间分辨率，以提供对细胞或组织内的叶绿体异质性的进一步了解。此外，该项目将培训下一代科学家，包括加州大学河滨大学未被充分代表的少数族裔学生，他们将在批判性思维、实验设计和执行以及科学交流方面进行培训。研究团队将通过出版物、会议演示和培训视频来传播这项赋能技术，以推动科学发现。这一奖项反映了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。

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.