The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning "empty." We used whole-genome sequencing of both mutants and molecular genetic analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (PRONE) catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpROP, the only ROP GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrates an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell-polarity formation and subsequent asymmetric cell division via activation of ROP signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.