Essential developmental processes in Physcomitrium patens require distinct levels of total activity provided by functionally redundant PpROP GTPases.

RHO (RAS homologous) GTPases regulate important cellular and developmental processes in most eukaryotes. Plant-specific ROP (RHO of plants) GTPase families expanded and functionally diversified during the evolution of vascular plants, but contain few members in nonvascular extant relatives of early land plants. Here, a systematic investigation of essential PpROP functions in the development of the nonvascular moss Physcomitrium patens is presented. This investigation was based on: knocking out individually or all possible combinations of each of the four PpROP genes, which encode nearly identical proteins; complementing knockout lines with wild-type (WT) or mutated PpROPs, or with heterologous homologs; and inducing PpROP overexpression. PpROPs were found to have previously unknown functions in cell proliferation, caulonema differentiation, and gametophore formation. PpROP functions were observed to display variable dependence on guanosine diphosphate (GDP)/guanosine triphosphate (GTP) cycling and to rely on distinct downstream signaling. Different cellular and developmental processes were determined to require distinct levels of total PpROP activity, rather than individual PpROPs. These observations provide important insights into PpROP functions and signaling in P. patens, enhancing our understanding of the evolution of the regulation of developmental processes by ROP/RHO GTPases. The evolutionary origin of the remarkable functional integration and sequence conservation within the PpROP family is discussed.