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Summary Many plants reproduce asexually by generating clonal progeny from vegetative tissues, a process known as vegetative reproduction. This reproduction mode contrasts with sexual reproduction, which enhances genetic diversity. The bryophyte Marchantia polymorpha L. adjusts its reproductive strategy in response to seasonal environmental cues, transitioning between vegetative and sexual reproduction. In this study, we identified a gene encoding the R2R3‐MYB transcription factor SHOT GLASS (MpSTG) as a critical regulator of gemma cup development. MpSTG was predominantly expressed in the gemma cup, apical notch, and sexual reproductive organs (gametangiophores). MpSTG mutation resulted in the formation of abnormal shot‐glass‐shaped structures lacking gemmae, which replaced functional gemma cups. Additionally, MpSTG‐disrupted plants failed to develop sexual reproductive organs, even under inductive conditions. In Arabidopsis thaliana, the MpSTG ortholog LATERAL ORGAN FUSION1 (AtLOF1) plays a pivotal role in lateral bud formation. We demonstrated that MpSTG can partially compensate for AtLOF1's function in lateral bud formation in A. thaliana. Our findings suggest that MpSTG is a key regulator of vegetative and sexual reproduction in M. polymorpha, and illustrate that evolutionarily conserved developmental mechanisms may function in both the gametophyte generation of bryophytes and the sporophyte generation of angiosperms.

ABSTRACT Rho/Rac of plant (ROP) GTPases are 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. The ROPGEF KARAPPO (MpKAR) is essential for gemma initiation, but 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 processes involving MpREN. Overexpression of MpROPGAP and 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, likely by controlling MpROP activation in M. polymorpha.

Abstract Auxin plays pleiotropic roles in plant development via gene regulation upon its perception by the receptors TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFBs). This auxin-regulated transcriptional control mechanism originated in the common ancestor of land plants. Although the complete loss of TIR1/AFBs causes embryonic lethality in Arabidopsis thaliana, it is unclear whether the requirement for TIR1-mediated auxin perception in cell viability can be generalized. The model liverwort Marchantia polymorpha has a minimal auxin signaling system with only a single TIR1/AFB, MpTIR1. Here we show by genetic, biochemical, and transcriptomic analyses that MpTIR1 functions as an evolutionarily conserved auxin receptor. Null mutants and conditionally knocked-out mutants of MpTIR1 were viable but incapable of forming any organs and grew as cell masses. Principal component analysis performed using transcriptomes at various developmental stages indicated that MpTIR1 is involved in the developmental transition from spores to organized thalli, during which apical notches containing stem cells are established. In Mptir1 cells, stem cell- and differentiation-related genes were up- and downregulated, respectively. Our findings suggest that, in M. polymorpha, auxin signaling is dispensable for cell division but is essential for three-dimensional patterning of the plant body by establishing pluripotent stem cells for organogenesis, a derived trait of land plants.

Abstract Cytokinin, a plant hormone, plays essential roles in organ growth and development. The type-B response regulator-mediated cytokinin signaling is repressed by type-A response regulators and is conserved in the liverwort Marchantia polymorpha. Its signal coordinates the development of diverse organs on the thallus body, such as the gemma cup, rhizoid, and air pores. Here we report that the type-B response regulator MpRRB upregulates the expression of the R2R3-MYB transcription factor GEMMA CUP-ASSOCIATED MYB1 (MpGCAM1) in M. polymorpha. Whereas both Mpgcam1 and Mprrb knockout mutants exhibited defects in gemma cup formation, the Mpgcam1 Mprra double mutant, in which cytokinin signaling is activated due to the lack of type-A response regulator, also formed no gemma cups. This suggests that MpGCAM1 functions downstream of cytokinin signaling. Inducible overexpression of MpGCAM1 produced undifferentiated cell clumps on the thalli of both wild-type and Mprrb. However, smaller thalli were formed in Mprrb compared to the wild-type after the cessation of overexpression. These results suggest that cytokinin signaling promotes gemma cup formation and cellular reprogramming through MpGCAM1, while cytokinin signals also participate in activating cell division during thallus development.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Phytohormone abscisic acid (ABA) plays a key role in stomata closure, osmostress acclimation, and vegetative and embryonic dormancy. Group B3 Raf protein kinases (B3-Rafs) serve as positive regulators of ABA and osmostress signaling in the moss Physcomitrium patens and the angiosperm Arabidopsis thaliana. While P. patens has a single B3-Raf called ARK, specific members of B3-Rafs among six paralogs regulate ABA and osmostress signaling in A. thaliana, indicating functional diversification of B3-Rafs in angiosperms. However, we found that the liverwort Marchantia polymorpha, belonging to another class of bryophytes, has three paralogs of B3-Rafs, MpARK1, MpARK2, and MpARK3, with structural variations in the regulatory domains of the polypeptides. By reporter assays of the P. patens ark line and analysis of genome-editing lines of M. polymorpha, we found that these B3-Rafs are functionally redundant in ABA response, with respect to inhibition of growth, tolerance to desiccation and expression of stress-associated transcripts, the majority of which are under the control of the PYR/PYL/RCAR-like receptor MpPYL1. Interestingly, gemmae in gemma cups were germinating only in mutant lines associated with MpARK1, indicating that dormancy in the gametophyte is controlled by a specific B3-Raf paralog. These results indicated not only conservation of the role of B3-Rafs in ABA and osmostress response in liverworts but also functional diversification of B3-Rafs, which is likely to have occurred in the early stages of land plant evolution.

Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana . Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis . In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.

Auxin plays critical roles in growth and development through the regulation of cell differentiation, cell expansion, and pattern formation. The auxin signal is mainly conveyed through a so-called nuclear auxin pathway involving the receptor TIR1/AFB, the transcriptional co-repressor AUX/IAA, and the transcription factor ARF with direct DNA-binding ability. Recent progress in sequence information and molecular genetics in basal plants has provided many insights into the evolutionary origin of the nuclear auxin pathway and its pleiotropic roles in land plant development. In this review, we summarize the latest knowledge of the nuclear auxin pathway gained from studies using basal plants, including charophycean green algae and two major model bryophytes, Marchantia polymorpha and Physcomitrella patens. In addition, we discuss the functional implication of the increase in genetic complexity of the nuclear auxin pathway during land plant evolution.

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss-and gain-of-function alleles for auxin synthesis-and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.

The phytohormone auxin plays a pivotal role in various developmental aspects in land plants. However, little is known of the auxin response and distribution in non-vascular plants. In this study, we made transgenic plants of the liverwort Marchantia polymorpha which express the uidA (GUS) reporter gene under control of the soybean auxin-inducible promoter, ProGH3, and used it to indirectly monitor auxin-mediated transcriptional activation in planta. Transgenic plants carrying ProGH3:GUS showed GUS activity in an auxin-dependent manner. Histochemical GUS staining was observed at the bottom of gemma cups in the process of vegetative propagation. Significant GUS activity was also detected around the gametophyte-sporophyte junction as well as the developing sporophyte after fertilization. These results suggest that the activity of auxin is crucial in both gametophyte and sporophyte development in M. polymorpha, and that the mechanism for auxin-mediated transcriptional activation had already been established when plants emerged on the terrestrial environment.