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.
