Land plants (embryophytes) are multicellular eukaryotes with a remarkable capacity to grow continuously during their life span. They achieve this by maintaining a reservoir of pluripotent stem cells in their meristems. In embryophytes, including both bryophytes and tracheophytes, the haploid gametophytic generation is inferred to be ancestrally free living. Gametophyte meristems are sites of auxin biosynthesis, but auxin promotes differentiation, and conversely, a lack of auxin results in the loss of differentiation. We report that in the liverwort Marchantia polymorpha, pluripotent stem cells are protected from auxin-mediated differentiation by the single B-class auxin response factor (MpARF2), an embryophyte-specific gene that antagonizes auxin responses. We describe the critical role of MpARF2 in meristem establishment and maintenance using reporter lines, loss- and gain-of-function alleles, and gene-interaction experiments. Mparf2 knockdown and knockout alleles, respectively, fail to maintain and form a functional meristem across developmental transitions, while overexpression of MpARF2 is sufficient to create additional meristems in dormant gemmae. Despite being an auxin-signaling antagonist, MpARF2 positively regulates auxin production by activating a YUCCA (MpYUC2) auxin biosynthetic enzyme in stem cells. Conversely, auxin responses mediated by the single A-ARF (MpARF1) antagonize both auxin production and MpARF2 expression in differentiating neighbor cells, creating a multicellular incoherent feedforward loop that limits meristem size. We propose that MpARF2 and auxin form a meristem organizer-modulating auxin insensitivity at the site of auxin production while providing signaling information to surrounding differentiating cells-and that this organizer was critical for the emergence of indeterminate growth in the ancestral embryophyte.

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.

In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha, the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW-AUXIN RESPONSIVE (MpLAXR), which is induced upon auxin reduction. Loss of MpLAXR function resulted in delayed cell cycle reactivation, and transient expression of MpLAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of MpLAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, MpLAXR is up-regulated to trigger cellular reprogramming. MpLAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNRÖSCHEN, which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants.