<i>pamiR</i> : INVESTIGATING PLANT CELLS ONE ORGANELLE AT A TIME

Abstract Functional genetic redundancy (FGR) within gene families limits the discovery of gene function in plants because single-gene perturbations often fail to produce informative phenotypes. Artificial microRNAs (amiRNAs) provide a strategy to silence multiple related genes simultaneously. However, the existing amiRNA-based libraries used for genetic gene function discovery in plants do not account for the subcellular localization of gene products, which can lead to pleiotropic or difficult-to-interpret phenotypes. Plastids are essential plant cell organelles that integrate central metabolic and signaling processes, including photosynthesis, hormone biosynthesis, and environmental responses. Here we introduce pamiR , a plastid-targeted amiRNA library designed to enable organelle-specific gene function discovery in Arabidopsis thaliana . Using plastid proteomic datasets, we identified high-confidence plastid-localized proteins and designed amiRNAs to target their gene(s) (families) minimizing FGR. This amiRNA library was introduced in a vector with fluorescence-accumulating seed technology enabling rapid, herbicide-free selection and screening in the first generation. Validation by next-generation sequencing, confirmed high representation and uniform distribution of amiRNAs within pamiR . Proof-of-concept screens recovered mutants affecting known and additional candidate genes involved in photosynthesis and abscisic acid biosynthesis. Therefore, the pamiR library provides a fast platform for plastid-focused genetic screens that is compatible with existing mutant collections. One-sentence summary The plastid amiRNA ( pamiR ) library enables organelle-specific forward genetics without functional genetic redundancy.