PhD Candidate University of California, Riverside Riverside, California
Body of Abstract: Plastids are DNA-containing signaling organelles that sense environmental stimuli to modulate their physiological and metabolic functions. In response to cell-type-specific developmental cues, they differentiate into specialized types to achieve goals that range from photosynthesis in chloroplasts to gravity sensing in amyloplasts. Both plastid signaling and differentiation are closely associated with regulation of the plastome. Although the general principles of plastid gene regulation at the transcriptional and post-transcriptional levels have been extensively illustrated at the organ and cell-type levels, our understanding of plastidic transcriptomics on the scale of an individual plastid remains rudimentary. The main challenge has been the lack of an easily accessible approach for routine transcript analysis of single plastids. Here we developed in-situ reverse transcription technology to visualize and quantify plastidic transcript abundance. This approach utilizes fluorescently labeled deoxyribonucleotide during in-situ reverse transcription to afford insight on transcriptional patterns within single plastids. Our technology, termed In-situ FLuorescence Analysis of Transcripts within Single Plastids (IFLATR-SP), allows a glimpse into the comparative abundance of various RNA species within individual plastids. As a proof of concept, we applied our method to quantify global and individual transcript levels within isolated chloroplasts when priming reverse transcription with either random or gene-specific primers, respectively. We expect that IFLATR-SP will enable investigations of a wide range of transcriptional and post-transcriptional gene regulatory events at the single plastid level.