Processes essential forPhyscomitrium patensprotonemal development require distinct levels of total activity provided by functionally redundant PpROP GTPases

RHO GTPases are key regulators of cellular and developmental processes in most eukaryotic organisms. ROPs (RHO of plants) constitute a plant-specific RHO subfamily. ROP families expanded and functionally diversified during the evolution of structurally complex vascular plants, but generally contain few members in non-vascular plants with ancient features. In vascular and non-vascular plants, ROP proteins are required for cell polarization, directional cell expansion, and mitotic cell plate positioning. All these processes are impaired by the disruption of PpROP activity in the non-vascular moss Physcomitrium patens . The aim of the study presented here was to further characterize PpROP functions during P. patens protonemal development by a) knocking out individually or in all possible combinations each of the four Rp ROP genes, which encode nearly identical proteins, b) complementing knock-out mutants with WT or mutant PpROP isoforms, or with heterologous homologs, and c) performing overexpression experiments. PpROPs were found to have additional previously unknown functions in the regulation of cell proliferation, caulonema differentiation, and gametophore formation. Furthermore, different cellular and developmental processes were shown to require distinct levels of total PpROP activity, rather than individual PpROP isoforms. Implications of the remarkable sequence conservation and functional integration within the PpROP protein family are discussed. One-sentence summary Knock-out, complementation, and overexpression experiments further defined PpROP functions in P. patens development, and demonstrated their dependence on distinct levels of total PpROP activity.