Regulation of ROP GTPase cycling between active/inactive states is essential for vegetative organogenesis in Marchantia polymorpha.

Rho/Rac of plant (ROP) GTPases are a plant-specific proteins that function as molecular switches, activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single copies of ROP (MpROP), GEFs (ROPGEF and SPIKE (SPK)), and GAPs (ROPGAP and ROP ENHANCER (REN)). MpROP regulates the development of various tissues and organs such as rhizoids, gemmae, and air chambers. While the ROPGEF, KARAPPO (MpKAR) is essential for gemma initiation, the functions of other ROP regulatory factors are less understood. This study focused on two GAPs: MpROPGAP and MpREN. Mpren single mutants showed defects in thallus growth, rhizoid tip growth, gemma development, and air chamber formation, whereas Mpropgap mutants showed no visible abnormalities. However, Mpropgap Mpren double mutants had more severe phenotypes than the Mpren single mutants, suggesting backup roles of MpROPGAP in MpREN-involving processes. Overexpression of MpROPGAP, MpREN resulted in similar gametophyte defects, highlighting the importance of MpROP activation/inactivation cycling (or balancing). Thus, MpREN predominantly, and MpROPGAP as a backup, regulate gametophyte development, most likely by controlling MpROP activation in M. polymorpha.