Two subtypes of GTPase-activating proteins coordinate tip growth and cell size regulation in Physcomitrium patens

The establishment of cell polarity is a prerequisite for many developmental processes. However, how it is achieved during tip growth in plants remains elusive. Here, we show that the RHO OF PLANTs (ROPs), ROP GUANINE NUCLEOTIDE EXCHANGE FACTORs (RopGEFs), and ROP GTPASE-ACTIVATING PROTEINs (RopGAPs) assemble into membrane domains in tip-growing cells of the moss Physcomitrium patens. The confinement of membrane domains requires redundant global inactivation of ROPs by PpRopGAPs and the PLECKSTRIN HOMOLOGY (PH) domain-containing RenGAP PpREN. Unexpectedly, PpRopGAPs and PpREN exert opposing effects on domain size and cell width upon overexpression. Biochemical and functional analyses indicate that PpRopGAPs are recruited to the membrane by active ROPs to restrict domain size through clustering, whereas PpREN rapidly inactivates ROPs and inhibits PpRopGAP-induced clustering. We propose that the activity- and clustering-based domain organization by RopGAPs and RenGAPs is a general mechanism for coordinating polarized cell growth and cell size regulation in plants. SignificanceUsing the moss Physcomitrium patens, we show that PpRopGAPs and PpRenGAP redundantly regulate tip growth by inactivating ROP GTPases and influencing their membrane organization. They display distinct GAP activities and binding capacities to ROPs and could oppositely regulate cell width when overexpressed. We propose that tip growth and cell size regulation are coordinated by two subtypes of GTPase-activating proteins that act together to maintain an optimal amount of active ROPs but reversely regulate the membrane clustering of ROPs. The involvement of functionally diverged GAPs in modulating ROP activity and localization may represent a general mechanism for cell morphology regulation in plants.