22 23 24 Manuscript SUMMARY 25 Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; 26 however, little is known about the molecular mechanisms underlying this process. 27 Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, 28 which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative 29 thallus [1]. The initial stage of gemma development involves elongation and asymmetric 30divisions of a specific type of epidermal cell, called a gemma initial, which forms on the 31 floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma 32 development, we focused on two allelic mutants in which no gemma initial formed; these 33 mutants were named karappo, meaning "empty". We used whole-genome sequencing of 34 both mutants, and molecular genetic analyses to identify the causal gene, KARAPPO (KAR), 35 which encodes a Rop guanine nucleotide exchange factor (RopGEF) carrying a PRONE 36 catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the 37 PRONE domain have significant GEF activity toward MpRop, the only Rop GTPase in M. 38 polymorpha. Moreover, genetic complementation experiments showed a significant role for 39 the N-and C-terminal variable regions in gemma development. Our investigation 40 demonstrated an essential role for KAR/RopGEF in the initiation of plantlet development 41 from a differentiated cell, which may involve cell polarity formation and subsequent 42 asymmetric cell division via activation of Rop signaling, implying a similar developmental 43 mechanism in vegetative reproduction of various land plants.44 45 KEYWORDS 46 asexual reproduction, small GTPase, cell polarity, evolution 47 48 3 RESULTS AND DISCUSSION 49 Gemma development in Marchantia polymorpha 50 Vegetative reproduction is a form of asexual reproduction in which clonal individuals 51 develop directly from vegetative tissues, such as leaves, stems, and roots. Vegetative 52 reproduction is a developmental process based on totipotency, which is the potential for a 53 cell, even a differentiated cell, to regenerate organs or whole plantlets [4-6]. Many plants in 54 diverse lineages exhibit vegetative reproduction, e.g. potato (Solanum tuberosum), which 55 produces tubers in underground stems, Kalanchoe diagremontiana, which forms plantlets 56 at the leaf margins, the Dhalia family, which develop root tubers, and the hen and chicken 57 fern (Asplennium bulbiferum), which grows small bulbils on the top of fronds [7]. However, 58 very little is known about the underlying molecular mechanisms of vegetative 59 reproduction. 60 One of the most basal lineages in extant land plants, the liverwort Marchantia 61 polymorpha, has the ability to propagate asexually by forming clonal plantlets, called 62 gemmae, in a cupule or "gemma cup", a cup-like receptacle formed on the dorsal side of 63 the thallus, which is the gametophyte plant body (Figure S1A). The development of the 64 gemma and gemma cup in M. pol...

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