pamiR: INVESTIGATING PLANT CELLS ONE ORGANELLE AT A TIME

Plant cell biochemistry is highly compartmentalized. Hence, forward genetic screening in an organelle-specific manner holds great potential to reveal novel loci controlling cell physiology. However, conventional forward genetics is a) limited by functional redundancy that can mask phenotypes in plant gene families and b) lacks organelle specificity. Here, we present the plastid-focused artificial microRNA (pamiR) resource designed to silence individual nuclear genes or co-targetable gene-family subsets encoding the organelle's proteome. The library contains 2,285 artificial microRNAs directed against 2,202 genes selected from a plastid-enriched, proteomics-supported target set. The constructs are expressed by a constitutive UBQ10 promoter in a binary vector carrying the FAST seed-fluorescence marker, allowing transformant selection before germination. Two proof-of-concept screens, one using photosynthetic phenotyping and one using paclobutrazol, a gibberellin biosynthesis inhibitor, recovered lines with phenotypes previously associated with simultaneous perturbation of multiple members of redundant plastid gene families such as light-harvesting complex b1 (LHCB1), K+ efflux antiporter (KEA), and 9-cis-epoxycarotenoid dioxygenase (NCED). We experimentally confirmed specific target depletion for selected candidates, including paralogue-resolved LHCB1 depletion by mass spectrometry, and provide predicted off-target annotations for all 2,285 guides. Untargeted LHCB family members remained unchanged, and across the proteins included in the proteome-wide differential-abundance analysis, no additional significantly decreased protein was detected. KEA1 immunoreactive signal also fell below the immunoblot detection limit in pamiRkea1/2 and an independent KEA1/KEA2 amiRNA line. Together, these data establish pamiR as a plastid-specific screening resource and provide a framework for prioritizing and independently validating candidate phenotypes.