Synthetic genomics involves the design, assembly, and transfer of artificially synthesized DNA fragments into target hosts to replace the native genome and construct viable forms of life. With advances in DNA synthesis and assembly techniques, the application of synthetic genomics in viruses, bacteria, and yeast has improved our knowledge of genome organization and function. Multicellular eukaryotic organisms are characterized by larger genomes, more complex epigenetic regulation, and widespread transposable elements, making genome synthesis challenging. Recently, the first synthetic multicellular eukaryotic organism was generated in the model plant Physcomitrium patens with a partially synthetic chromosome arm. Here, we introduce the design and assembly principles of moss genome synthesis. We also discuss the remaining technical barriers in the application of synthetic genomics in seed plants.

The CLAVATA pathway plays a key role in the regulation of multicellular shoot and root meristems in flowering plants. In Arabidopsis, CLAVATA 3-like signaling peptides (CLEs) act via receptor-like kinases CLAVATA 1 and CRINKLY 4 (CR4). In the moss Physcomitrium patens, PpCLAVATA and PpCR4 were previously studied independently and shown to play conserved roles in the regulation of cell proliferation and differentiation. The plant calpain DEFECTIVE KERNEL 1 (DEK1) has been identified as another key regulator of cell division and cell fate in vascular plants and bryophytes. The functional interaction between CLAVATA, CR4, and DEK1 remains unknown. Here, we show that P. patens crinkly4 and dek1 mutants respond differently to CLE peptide treatments suggesting their distinct roles in the CLAVATA pathway. Reduced CLAVATA-mediated suppression of leafy shoot growth in Δcr4 mutants indicates that PpCR4 is involved in CLV3p perception, most likely as a receptor. The CLV3p strongly suppressed leaf vein development in Δcr4 mutants, suggesting that other receptors are involved in these processes and indicating a potential role of PpCR4 in organ sensitization to CLEs.

The conquest of land posed severe problems to plants which they had to cope with by adapting biosynthetic capacities. Adaptations to respond to UV irradiation, water loss, pathogen and herbivore defense, and the earth\'s pull were essential. Chemical compounds alleviating these problems can be synthesized by the phenylpropanoid pathway, the core of which are three enzymes: phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase, and 4-coumaric acid coenzyme A-ligase (4CL). The genomes of model organisms, Chara braunii as aquatic alga and the two bryophytes Physcomitrium patens and Marchantia polymorpha, were searched for sequences encoding PAL and 4CL and selected sequences heterologously expressed in Escherichia coli for biochemical characterization. Several possible isoforms were identified for both enzymes in Marchantia polymorpha and Physcomitrium patens, while only one or two isoforms could be retrieved for Chara braunii. Active forms of both enzymes were found in all three organisms, although the catalytic efficiencies varied in a wide range. l-Phenylalanine was accepted as best substrate by all PAL-like enzymes, despite annotations in some cases suggesting different activities. The substrate spectrum of 4CLs was more diverse, but caffeic and/or 4-coumaric acids generally were the best-accepted substrates. Our investigations show that PAL and 4CL, important enzymes for the formation of phenolic compounds, are present and active in extant charophytes and bryophytes as model organisms for the conquest of land.

The transition from two-dimensional (2D) to 3D growth likely facilitated plants to colonize land, but its heterogeneity is not well understood. In this study, we utilized single-cell RNA sequencing to analyze the moss Physcomitrium patens, whose morphogenesis involves a transition from 2D to 3D growth. We profiled over 17,000 single cells covering all major vegetative tissues, including 2D filaments (chloronema and caulonema) and 3D structures (bud and gametophore). Pseudotime analyses revealed larger numbers of candidate genes that determine cell fates for 2D tip elongation or 3D bud differentiation. Using weighted gene co-expression network analysis, we identified a module that connects β-type carbonic anhydrases (βCAs) with auxin. We further validated the cellular expression patterns of βCAs and demonstrated their roles in 3D gametophore development. Overall, our study provides insights into cellular heterogeneity in a moss and identifies molecular signatures that underpin the 2D-to-3D growth transition at single-cell resolution.

The study of moss calyptra form and function began almost 250 years ago, but calyptra research has remained a niche endeavor focusing on only a small number of species. Recent advances have focused on calyptra cuticular waxes, which function in dehydration protection of the immature sporophyte apex. The physical presence of the calyptra also plays a role in sporophyte development, potentially via its influence on auxin transport. Progress developing genomic resources for mosses beyond the model Physcomitrium patens, specifically for species with larger calyptrae and taller sporophytes, in combination with advances in CRISPR-Cas9 genome editing will enable the influence of the calyptra on gene expression and the production of RNAs and proteins that coordinate sporophyte development to be explored.

Moss plants appear in the early stages of land colonization and possess varying degrees of dehydration tolerance. In this study, a protein called PpFAS1.3 was identified, which contains a fasciclin 1-like domain and is essential for the moss Physcomitrium patens\' response to short-term rapid dehydration. When the FAS1.3 protein was knocked out, leafyshoots showed a significant decrease in tolerance to rapid dehydration, resulting in accelerated water loss and increased membrane leakage. Phylogenetic analysis suggests that PpFAS1.3 and its homologous proteins may have originated from bacteria and are specifically found in non-vascular plants like mosses and liverworts. As a dehydration-related protein, FAS1.3 plays a significant role in regulating lipid metabolism, particularly in the synthesis of free fatty acids (FFA) and the metabolism of two phospholipids, PC and PA. This discovery highlights the close connection between PpFAS1.3 and lipid metabolism, providing new insights into the molecular mechanisms underlying plant adaptation to stresses.

Plant movements for survival are nontrivial. Antheridia in the moss Physcomitrium patens (P. patens) use motion to eject sperm in the presence of water. However, the biological and mechanical mechanisms that actuate the process are unknown. Here, the burst of the antheridium of P. patens, triggered by water, results from elastic instability and is determined by an asymmetric change in cell geometry. The tension generated in jacket cell walls of antheridium arises from turgor pressure, and is further promoted when the inner walls of apex burst in hydration, causing water and cellular contents of apex quickly influx into sperm chamber. The outer walls of the jacket cells are strengthened by NAC transcription factor VNS4 and serve as key morphomechanical innovations to store hydrostatic energy in a confined space in P. patens. However, the antheridium in liverwort Marchantia polymorpha (M. polymorpha) adopts a different strategy for sperm release; like jacket cell outer walls of P. patens, the cells surrounding the antheridium of M. polymorpha appear to play a similar role in the storage of energy. Collectively, the work shows that plants have evolved different ingenious devices for sperm discharge and that morphological innovations can differ.

Here, we report our approach to peptidomic analysis of the plant model Physcomitrium patens. Intracellular and extracellular peptides were extracted under conditions preventing proteolytic digestion by endogenous proteases. The extracts were fractionated on size exclusion columns to isolate intracellular peptides and on reversed-phase cartridges to isolate extracellular peptides, with the isolated peptides subjected to LC-MS/MS analysis. Mass spectrometry data were analyzed for the presence of peptides derived from the known proteins or microproteins encoded by small open reading frames (<100 aa, smORFs) predicted in the moss genome. Experimental details are provided for each step.

In plants, asymmetric cell divisions result in distinct cell fates forming large and small daughter cells, adding to the cellular diversity in an organ. SCARECROW (SCR), a GRAS domain-containing transcription factor controls asymmetric periclinal cell divisions in flowering plants by governing radial patterning of ground tissue in roots and cell proliferation in leaves. Though SCR homologs are present across land plant lineages, the current understanding of their role in cellular patterning and leaf development is mostly limited to flowering plants. Our phylogenetic analysis identified three SCR homologs in moss Physcomitrium patens, amongst which PpSCR1 showed highest expression in gametophores and its promoter activity was prominent at the mid-vein and the flanking leaf blade cells pointing towards its role in leaf development. Notably, out of the three SCR homologs, only the ppscr1 knock-out lines developed slender leaves with four times narrower leaf blade and three times thicker mid-vein. Detailed histology studies revealed that slender leaf phenotype is either due to the loss of anticlinal cell divisions or failure of periclinal division suppression in the leaf blade. RNA-Seq analyses revealed that genes responsible for cell division and differentiation are expressed differentially in the mutant. PpSCR1 overexpression lines exhibited significantly wider leaf lamina, further reconfirming the role in leaf development. Together, our data suggests that PpSCR1 is involved in the leaf blade and mid-vein development of moss and that its role in the regulation of cell division and proliferation is ancient and conserved among flowering plants and mosses.

Abscisic acid (ABA)-mediated abiotic stress tolerance causes plant growth inhibition. Under such stress conditions, some mosses generate de novo stress-resistant stem cells, also called brood cells or brachycytes, that do not exist under normal conditions. However, the cell physiological basis of the growth inhibition and the stem cell formation is not well understood. Here, we show that the ABA-induced growth inhibition of the moss Physcomitrium patens apical protonemal cells (protonemal stem cells) is mediated through a shift from asymmetric to symmetric cell division. This change of the cell division mode, and consequently change of stem cell activity, is substantiated by dampening cell polarity and cell proliferative activity through the altered distribution of cytoskeletal elements, the mitotic spindle and the vacuole, which results in the production of stress-resistant stem cells. Alteration of the cell physiological data is supported by the results of RNAseq analysis indicating rapid changes in both cell polarity and cell cycle regulation, while long-term treatments with ABA for 5 to 10 days impact mainly the transcriptional and translational regulation. The regulation of cell polarity and cell cycle genes suggests growth arrest mediated by small GTPases (ROPs) and their guanine exchange factors (ROPGEFs) and by cyclin and cyclin-dependent-kinase complex, respectively. Our data suggest that a tradeoff relationship between growth ability and abiotic stress response in the moss is substantiated by ABA signaling to suppress cell polarity and asymmetric cell growth and may play a pivotal role in stem cell fate conversion to newly produced stress-resistant stem cells.