DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

SUPPRESSOR OF MAX2 1-LIKE (SMXL) homologs are MAX2-dependent repressors of Physcomitrium patens growth.

SUPPRESSOR OF MAX2 (SMAX)1-LIKE (SMXL) proteins are a plant-specific clade of type I HSP100/Clp-ATPases. SMXL genes are present in virtually all land plant genomes. However, they have mainly been studied in angiosperms. In Arabidopsis (Arabidopsis thaliana), 3 functional SMXL subclades have been identified: SMAX1/SMXL2, SMXL345, and SMXL678. Of these, 2 subclades ensure endogenous phytohormone signal transduction. SMAX1/SMXL2 proteins are involved in KAI2 ligand (KL) signaling, while SMXL678 proteins are involved in strigolactone (SL) signaling. Many questions remain regarding the mode of action of these proteins, as well as their ancestral roles. We addressed these questions by investigating the functions of the 4 SMXL genes in the moss Physcomitrium patens. We demonstrate that PpSMXL proteins are involved in the conserved ancestral MAX2-dependent KL signaling pathway and negatively regulate growth. However, PpSMXL proteins expressed in Arabidopsis cannot replace SMAX1 or SMXL2 function in KL signaling, whereas they can functionally replace SMXL4 and SMXL5 and restore root growth. Therefore, the molecular functions of SMXL proteins are conserved, but their interaction networks are not. Moreover, the PpSMXLC/D clade positively regulates SL signal transduction in P. patens. Overall, our data reveal that SMXL proteins in moss mediate crosstalk between the SL and KL signaling pathways.

The bryophytes Physcomitrium patens and Marchantia polymorpha as model systems for studying evolutionary cell and developmental biology in plants.

Bryophytes are nonvascular spore-forming plants. Unlike in flowering plants, the gametophyte (haploid) generation of bryophytes dominates the sporophyte (diploid) generation. A comparison of bryophytes with flowering plants allows us to answer some fundamental questions raised in evolutionary cell and developmental biology. The moss Physcomitrium patens was the first bryophyte with a sequenced genome. Many cell and developmental studies have been conducted in this species using gene targeting by homologous recombination. The liverwort Marchantia polymorpha has recently emerged as an excellent model system with low genomic redundancy in most of its regulatory pathways. With the development of molecular genetic tools such as efficient genome editing, both P. patens and M. polymorpha have provided many valuable insights. Here, we review these advances with a special focus on polarity formation at the cell and tissue levels. We examine current knowledge regarding the cellular mechanisms of polarized cell elongation and cell division, including symmetric and asymmetric cell division. We also examine the role of polar auxin transport in mosses and liverworts. Finally, we discuss the future of evolutionary cell and developmental biological studies in plants.

Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function.

MAIN CONCLUSION: Developing bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms. All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling.

Myosin XI, a model of its conserved role in plant cell tip growth.

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.

Ethylene controls three-dimensional growth involving reduced auxin levels in the moss Physcomitrium patens.

The conquest of land by plants was concomitant with, and possibly enabled by, the evolution of three-dimensional (3D) growth. The moss Physcomitrium patens provides a model system for elucidating molecular mechanisms in the initiation of 3D growth. Here, we investigate whether the phytohormone ethylene, which is believed to have been a signal before land plant emergence, plays a role in 3D growth regulation in P. patens. We report ethylene controls 3D gametophore formation, based on results from exogenously applied ethylene and genetic manipulation of PpEIN2, which is a central component in the ethylene signaling pathway. Overexpression (OE) of PpEIN2 activates ethylene responses and leads to earlier formation of gametophores with fewer gametophores produced thereafter, phenocopying ethylene-treated wild-type. Conversely, Ppein2 knockout mutants, which are ethylene insensitive, show initially delayed gametophore formation with more gametophores produced later. Furthermore, pharmacological and biochemical analyses reveal auxin levels are decreased in the OE lines but increased in the knockout mutants. Our results suggest that evolutionarily, ethylene and auxin molecular networks were recruited to build the plant body plan in ancestral land plants. This might have played a role in enabling ancient plants to acclimate to the continental surfaces of the planet.

VAPYRIN-like is required for development of the moss Physcomitrella patens.

The VAPYRIN (VPY) gene in Medicago truncatula and Petunia hybrida is required for arbuscular mycorrhizal (AM) symbiosis. The moss Physcomitrella patens has a close homolog (VPY-like, VPYL), although it does not form AM. Here, we explore the phylogeny of VPY and VPYL in land plants, and study the expression and developmental function of VPYL in Ppatens We show that VPYL is expressed primarily in the protonema, the early filamentous stage of moss development, and later in rhizoids arising from the leafy gametophores and in adult phyllids. Knockout mutants have specific phenotypes in branching of the protonema and in cell division of the leaves (phyllids) in gametophores. The mutants are responsive to auxin and strigolactone, which are involved in regulation of protonemal branching, indicating that hormonal signaling in the mutants is not affected in hormonal signaling. Taken together, these results suggest that VPYL exerts negative regulation of protonemal branching and cell division in phyllids. We discuss VPY and VPYL phylogeny and function in land plants in the context of AM symbiosis in angiosperms and development in the moss.

A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for This GTPase in Subcellular and Tissue-Level Patterning.

Rho of Plants (ROPs) are GTPases that regulate polarity and patterned wall deposition in plants. As these small, globular proteins have many interactors, it has been difficult to ensure that methods to visualize ROP in live cells do not affect ROP function. Here, motivated by work in fission yeast (Schizosaccharomyces pombe), we generated a fluorescent moss (Physcomitrium [Physcomitrella] patens) ROP4 fusion protein by inserting mNeonGreen after Gly-134. Plants harboring tagged ROP4 and no other ROP genes were phenotypically normal. Plants lacking all four ROP genes comprised an unpatterned clump of spherical cells that were unable to form gametophores, demonstrating that ROP is essentially for spatial patterning at the cellular and tissue levels. The functional ROP fusion protein formed a steep gradient at the apical plasma membranes of growing tip cells. ROP also predicted the site of branch formation in the apical cell at the onset of mitosis, which occurs one to two cell cycles before a branch cell emerges. While fluorescence recovery after photobleaching studies demonstrated that ROP dynamics do not depend on the cytoskeleton, acute depolymerization of the cytoskeleton removed ROP from the membrane only in recently divided cells, pointing to a feedback mechanism between the cell cycle, cytoskeleton, and ROP.

SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens).

Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P. patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.

Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens.

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio-lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1-1 and 1-2). Both PpAN1 genes complemented the A. thaliana an-1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1-promoter- uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1-1 and PpAN1-2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip-growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α-tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1-1/1-2 double-knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism.

Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired gravitropism to sense gravity and change their growth direction. In addition, plants also adjust their morphogenesis in response to different gravitational forces in a phenomenon known as gravity resistance. However, the gravity resistance phenomenon in plants is poorly understood due to the prevalence of 1 g gravitational force on Earth: not only it is difficult to culture plants at gravity > 1 g(hypergravity) for a long period of time but it is also impossible to create a < 1 genvironment (µg, micro g) on Earth without specialized facilities. Despite these technical challenges, it is important to understand how plants grow in different gravity conditions in order to understand land plant adaptation to the 1 g environment or for outer space exploration. To address this, we have developed a centrifugal device for a prolonged duration of plant culture in hypergravity conditions, and a project to grow plants under the µg environment in the International Space Station is also underway. Our plant material of choice is Physcomitrium (Physcomitrella) patens, one of the pioneer plants on land and a model bryophyte often used in plant biology. In this review, we summarize our latest findings regarding P. patens growth response to hypergravity, with reference to our on-going "Space moss" project. In our ground-based hypergravity experiments, we analyzed the morphological and physiological changes and found unexpected increments of chloroplast size and photosynthesis rate, which might underlie the enhancement of growth and increase in the number of gametophores and rhizoids. We further discussed our approaches at the cellular level and compare the gravity resistance in mosses and that in angiosperms. Finally, we highlight the advantages and perspectives from the space experiments and conclude that research with bryophytes is beneficial to comprehensively and precisely understand gravitational responses in plants.

PpGRAS12 acts as a positive regulator of meristem formation in Physcomitrium patens.

KEY MESSAGE: This study focused on the key regulatory function of Physcomitrium patens GRAS12 gene underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life. The miR171-GRAS module has been identified as a key player in meristem maintenance in angiosperms. PpGRAS12 is a member of the GRAS family and a validated target for miR171 in Physcomitrium (Physcomitrella) patens. Here we show a regulatory function of miR171 at the gametophytic vegetative growth stage and targeted deletion of the PpGRAS12 gene adversely affects sporophyte production since fewer sporophytes were produced in ΔPpGRAS12 knockout lines compared to wild type moss. Furthermore, highly specific and distinct growth arrests were observed in inducible PpGRAS12 overexpression lines at the protonema stage. Prominent phenotypic aberrations including the formation of multiple apical meristems at the gametophytic vegetative stage in response to elevated PpGRAS12 transcript levels were discovered via scanning electron microscopy. The production of multiple buds in the PpGRAS12 overexpression lines similar to ΔPpCLV1a/1b disruption mutants is accompanied by an upregulation of PpCLE and downregulation of PpCLV1, PpAPB, PpNOG1, PpDEK1, PpRPK2 suggesting that PpGRAS12 acts upstream of these genes and negatively regulates the proposed pathway to specify simplex meristem formation. As CLV signaling pathway components are not present in the chlorophytic or charophytic algae and arose with the earliest land plants, we identified a key regulatory function of PpGRAS12 underlying an increasing plant complexity, an important step in plant terrestrialization and the evolutionary history of life.

Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts.

KEY MESSAGE: Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood. Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems' antenna complexes. Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes. In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.

Fundamental mechanisms of the stem cell regulation in land plants: lesson from shoot apical cells in bryophytes.

KEY MESSAGE: This review compares the molecular mechanisms of stem cell control in the shoot apical meristems of mosses and angiosperms and reveals the conserved features and evolution of plant stem cells. The establishment and maintenance of pluripotent stem cells in the shoot apical meristem (SAM) are key developmental processes in land plants including the most basal, bryophytes. Bryophytes, such as Physcomitrium (Physcomitrella) patens and Marchantia polymorpha, are emerging as attractive model species to study the conserved features and evolutionary processes in the mechanisms controlling stem cells. Recent studies using these model bryophyte species have started to uncover the similarities and differences in stem cell regulation between bryophytes and angiosperms. In this review, we summarize findings on stem cell function and its regulation focusing on different aspects including hormonal, genetic, and epigenetic control. Stem cell regulation through auxin, cytokinin, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling and chromatin modification by Polycomb Repressive Complex 2 (PRC2) and PRC1 is well conserved. Several transcription factors crucial for SAM regulation in angiosperms are not involved in the regulation of the SAM in mosses, but similarities also exist. These findings provide insights into the evolutionary trajectory of the SAM and the fundamental mechanisms involved in stem cell regulation that are conserved across land plants.

Rab-E and its interaction with myosin XI are essential for polarised cell growth.

The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.

Simultaneous imaging and functional studies reveal a tight correlation between calcium and actin networks.

Tip-growing cells elongate in a highly polarized manner via focused secretion of flexible cell-wall material. Calcium has been implicated as a vital factor in regulating the deposition of cell-wall material. However, deciphering the molecular and mechanistic calcium targets in vivo has remained challenging. Here, we investigated intracellular calcium dynamics in the moss Physcomitrella patens, which provides a system with an abundant source of genetically identical tip-growing cells, excellent cytology, and a large molecular genetic tool kit. To visualize calcium we used a genetically encoded cytosolic FRET probe, revealing a fluctuating tipward gradient with a complex oscillatory profile. Wavelet analysis coupled with a signal-sifting algorithm enabled the quantitative comparison of the calcium behavior in cells where growth was inhibited mechanically, pharmacologically, or genetically. We found that cells with suppressed growth have calcium oscillatory profiles with longer frequencies, suggesting that there is a feedback between the calcium gradient and growth. To investigate the mechanistic basis for this feedback we simultaneously imaged cytosolic calcium and actin, which has been shown to be essential for tip growth. We found that high cytosolic calcium promotes disassembly of a tip-focused actin spot, while low calcium promotes assembly. In support of this, abolishing the calcium gradient resulted in dramatic actin accumulation at the tip. Together these data demonstrate that tipward calcium is quantitatively linked to actin accumulation in vivo and that the moss P. patens provides a powerful system to uncover mechanistic links between calcium, actin, and growth.

Physcomitrium patens: A Single Model to Study Oriented Cell Divisions in 1D to 3D Patterning.

Development in multicellular organisms relies on cell proliferation and specialization. In plants, both these processes critically depend on the spatial organization of cells within a tissue. Owing to an absence of significant cellular migration, the relative position of plant cells is virtually made permanent at the moment of division. Therefore, in numerous plant developmental contexts, the (divergent) developmental trajectories of daughter cells are dependent on division plane positioning in the parental cell. Prior to and throughout division, specific cellular processes inform, establish and execute division plane control. For studying these facets of division plane control, the moss Physcomitrium (Physcomitrella) patens has emerged as a suitable model system. Developmental progression in this organism starts out simple and transitions towards a body plan with a three-dimensional structure. The transition is accompanied by a series of divisions where cell fate transitions and division plane positioning go hand in hand. These divisions are experimentally highly tractable and accessible. In this review, we will highlight recently uncovered mechanisms, including polarity protein complexes and cytoskeletal structures, and transcriptional regulators, that are required for 1D to 3D body plan formation.

Functional characterization of LIKE HETEROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens: its conserved protein interactions in land plants.

In flowering plants, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2 (TFL2) is known to interact with polycomb group (PcG) and non-PcG proteins and control developmental programs. LHP1/TFL2 is an ancient protein and has been characterized in the early-divergent plant Physcomitrella patens. However, interacting partners of PpLHP1 other than the chromomethylase PpCMT have not been identified to date. Also, while functional polycomb repressive complex 2 (PRC2) is known to exist in P. patens, there is no experimental evidence to support the existence of PRC1-like complexes in these mosses. In this study, using protein-protein interaction methods, transient expression assays and targeted gene knockout strategy, we report the conserved properties of LHP1/TFL2 using the Physcomitrella system. We show that a PRC1-like core complex comprising of PpLHP1 and the putative PRC1 Really Interesting New Gene (RING)-finger proteins can form in vivo. Also, the interaction between PpRING and the PRC2 subunit PpCLF further sheds light on the possible existence of combinatorial interactions between the Polycomb Repressive Complex (PRC) in early land plants. Based on the interaction between PpLHP1 and putative hnRNP PpLIF2-like in planta, we propose that the link between PpLHP1 regulation and RNA metabolic processes was established early in plants. The conserved subnuclear distribution pattern of PpLHP1 in moss protonema further provides insight into the manner in which LHP1/TFL2 are sequestered in the nucleoplasm in discrete foci. The PpLHP1 loss-of-function plants generated in this study share some of the pleiotropic defects with multiple aberrations reported in lhp1/tfl2. Taken together, this work documents an active role for PpLHP1 in epigenetic regulatory network in P. patens.