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

Rho/Rac of plant (ROP) GTPases are a plant-specific subfamily of Rho small GTP-binding proteins that function as molecular switches by being converted to the active state by guanine nucleotide exchange factors (GEFs) and to the inactive state by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single-copy genes encoding ROP (MpROP), two types of GEFs (ROPGEF and SPIKE (SPK)), and two types of GAPs (ROPGAP and ROP enhancer (REN)). MpROP regulates the development of various organs, including the air chambers, rhizoids, and clonal propagule gemmae. While the sole PRONE-type ROPGEF, KARAPPO (MpKAR), plays an essential role in gemma initiation, little is known about the in-planta functions of other ROP regulatory factors in M. polymorpha . In this study, we focused on the functions of two types of GAPs: MpROPGAP and MpREN. Loss-of-function Mp ren ge single mutants showed pleiotropic defects in thallus growth, air chamber formation, rhizoid tip growth, and gemma development, whereas MpROPGAP mutants showed no detectable abnormalities. Despite the distinctive domain structures of MpROPGAP and MpREN, Mp ropgap ge Mp ren ge double mutants showed more severe phenotypes than the Mp ren ge single mutants, suggesting redundant functions of MpROPGAP and MpREN in gametophyte organogenesis. Interestingly, overexpression of Mp ROPGAP , Mp REN , and dominant-negative Mp ROP (Mp ROP DN ) resulted in similar air chamber defects, as well as loss-of-function of Mp REN and Mp ROPGAP and overexpression of constitutively active Mp ROP (Mp ROP CA ), suggesting importance of activation/inactivation cycling (or balancing) of MpROP. Furthermore, we proved the contributions of the sole DOCK family GEF, MpSPK, to MpROP-regulated air chamber formation. In summary, our results demonstrate a significant role of the two GAPs in the development of various organs and that the two GEFs are responsible for organogenesis through the control of the MpROP active/inactive cycle in the vegetative growth of M. polymorpha .