The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.

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.

Intracellular and Extracellular Peptidomes of the Model Plant, Physcomitrium patens.

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.

Characterization of the fiber-like cortical cells in moss gametophytes.

MAIN CONCLUSION: Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-ß-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-ß-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients.

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss-cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Drought responses and population differentiation of Calohypnum plumiforme inferred from comparative transcriptome analysis.

Bryophytes, known as poikilohydric plants, possess vegetative desiccation-tolerant (DT) ability to withstand water deficit stress. Consequently, they offer valuable genetic resources for enhancing resistance to water scarcity stress. In this research, we examined the physiological, phytohormonal, and transcriptomic changes in DT mosses Calohypnum plumiforme from two populations, with and without desiccation treatment. Comparative analysis revealed population differentiation at physiological, gene sequence, and expression levels. Under desiccation stress, the activities of superoxide dismutase (SOD) and peroxidase (POD) showed significant increases, along with elevation of soluble sugars and proteins, consistent with the transcriptome changes. Notable activation of the bypass pathway of JA biosynthesis suggested their roles in compensating for JA accumulation. Furthermore, our analysis revealed significant correlations among phytohormones and DEGs in their respective signaling pathway, indicating potential complex interplays of hormones in C plumiforme. Protein phosphatase 2C (PP2C) in the abscisic acid signaling pathway emerged as the pivotal hub in the phytohormone crosstalk regulation network. Overall, this study was one of the first comprehensive transcriptome analyses of moss C. plumiforme under slow desiccation rates, expanding our knowledge of bryophyte transcriptomes and shedding light on the gene regulatory network involved in response to desiccation, as well as the evolutionary processes of local adaptation across moss populations.

Evaluation of Orthotrichum lyellii moss as a biomonitor of diesel exhaust.

Exhaust from diesel combustion engines is an important contributor to urban air pollution and poses significant risk to human health. Diesel exhaust contains a chemical class known as nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) and is enriched in 1-nitropyrene (1-NP), which has the potential to serve as a marker of diesel exhaust. The isomeric nitro-PAHs 2-nitropyrene (2-NP) and 2-nitrofluoranthene (2-NFL) are secondary pollutants arising from photochemical oxidation of pyrene and fluoranthene, respectively. Like other important air toxics, there is not extensive monitoring of nitro-PAHs, leading to gaps in knowledge about relative exposures and urban hotspots. Epiphytic moss absorbs water, nutrients, and pollutants from the atmosphere and may hold potential as an effective biomonitor for nitro-PAHs. In this study we investigate the suitability of Orthotrichum lyellii as a biomonitor of diesel exhaust by analyzing samples of the moss for 1-NP, 2-NP, and 2-NFL in the Seattle, WA metropolitan area. Samples were collected from rural parks, urban parks, residential, and commercial/industrial areas (N = 22 locations) and exhibited increasing concentrations across these land types. Sampling and laboratory method performance varied by nitro-PAH, but was generally good. We observed moderate to moderately strong correlation between 1-NP and select geographic variables, including summer normalized difference vegetation index (NDVI) within 250 m (r = -0.88, R2 = 0.77), percent impervious surface within 50 m (r = 0.83, R2 = 0.70), percent high development land use within 500 m (r = 0.77, R2 = 0.60), and distance to nearest secondary and connecting road (r = -0.75, R2 = 0.56). The relationships between 2-NP and 2-NFL and the geographic variables were generally weaker. Our results suggest O. lyellii is a promising biomonitor of diesel exhaust, specifically for 1-NP. To our knowledge this pilot study is the first to evaluate using moss concentrations of nitro-PAHs as biomonitors of diesel exhaust.

PpSCARECROW1 (PpSCR1) regulates leaf blade and mid-vein development in Physcomitrium patens.

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.

Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant.

KEY MESSAGE: To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.

First step taken toward artificial plant genome.

Scientists make partially synthetic version of moss chromosome, aiming to harness plant for industry.

Removal of potentially toxic elements from water with the moss-tufa micro-filtration system.

Mosses are an integral component in the tufa sedimentary landscape. In this study, we investigated the use of the porous moss-tufa structure as a filtration system for removing potentially toxic elements (PTEs) from water samples. Three species of mosses that commonly grow on tufa were selected, and the PTEs filtered by the moss-tufa system were identified by inductively coupled plasma mass spectrometry. The bioconcentration factor (BCF) of mosses was calculated to compare the enrichment effects of different mosses on PTEs. Likewise, the level of PTEs flowing through the moss-tufa system was measured, and the water quality removal rate (C) was calculated accordingly. The results revealed that the moss-tufa system was mainly composed of Fissidens grandifrons Brid., Hydrogonium dixonianum P. C. Chen, and Cratoneuron filicinum (Hedw.) Spruce var. filicinum. Among these, Fissidens grandifrons Brid. reported the highest retention capacity for PTEs. Collectively, the moss-tufa filtration system displayed a strong retention capacity and removal rate of Mn, Pb, and Ni from the water sample. The removal of PTEs by the moss-tufa system was mainly based on the enrichment of mosses and the adsorption-retention ability of tufa. In conclusion, the moss-tufa micro-filtration system displayed the effective removal of PTEs from water samples and could be applied to control the levels of toxic elements in karst water bodies.

A designer synthetic chromosome fragment functions in moss.

Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella.

KEY MESSAGE: Characterization of Physcomitrella 3'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long-standing interaction among land plants and fungi.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

Enhancement of salt tolerance of alfalfa: Physiological and molecular responses of transgenic alfalfa plants expressing Syntrichia caninervis-derived ScABI3.

Alfalfa (Medicago sativa L.), a perennial forage plant, is a rich source of nutrients such as vitamins, minerals, and proteins. Salt stress, however, impedes its growth. The plant-specific transcription factor abscisic acid insensitive 3 (ABI3) has a critical contribution to the control of abscisic acid (ABA) signaling pathway and abiotic stress response. The gene ScABI3 from Syntrichia caninervis, a moss species tolerant to desiccation, could be considered a potential candidate gene to modify alfalfa's nutritional and growth aspects. However, it remains unclear how ScABI3 affects the salt stress response of transgenic alfalfa. Therefore, we elucidated the role and molecular mechanism of ScABI3 from S. caninervis as an ABA signaling factor in transgenic alfalfa. Our findings demonstrate that ScABI3 overexpression in transgenic alfalfa improves salt tolerance by promoting relative water content, antioxidant enzyme activity, and photosynthetic parameters. Furthermore, the key genes of plant hormone signaling and the classical salt tolerance pathway were activated in ScABI3 transgenic lines under salt stress. Based on these results, ScABI3 could be considered a potentially critical candidate gene to alleviate salt stress in alfalfa. The present study provides valuable insights for developing transgenic crop breeding strategies for saline-alkaline soils.

Effects of fluorescent tags and activity status on the membrane localization of ROP GTPases.

Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.

The threat of groundwater pollution for petrifying springs; defining nutrient threshold values for an endangered bryophyte dominated habitat.

Eutrophication by human activities is increasingly affecting ecosystem functioning and plant community composition. So far, studies mainly focus on the effects of atmospheric nitrogen deposition, surface water eutrophication or soil nutrient accumulation. Groundwater pollution of spring habitats, however, has received much less attention, although numerous papers report groundwater nutrient enrichment worldwide. This study presents a survey on groundwater pollution (with emphasis on nitrate and phosphate) and bryophyte composition in 51 ambient petrifying springs in 5 NW European countries, which were compared to published data from 173 other sites in 11 European countries. The reviewed dataset covers a broad range of unpolluted to heavily polluted springs with nitrate concentrations between 0.7 and 3227 µmol l-1. Most petrifying springs in the rural lowlands of NW Europe were found to have elevated concentrations of nitrate and phosphate with the most polluted springs occurring in The Netherlands. The cover of individual characteristic bryophyte species significantly correlates with groundwater nutrient concentrations indicating that nutrient pollution of spring waters affects bryophyte composition. Palustriella commutata, Eucladium verticillatum and Brachythecium rivulare prefer unpolluted petrifying springs whereas Cratoneuron filicinum and Pellia endiviifolia show a much broader tolerance to groundwater pollution. In order to sustain at least the basic conditions for the typical bryophyte composition of petrifying springs habitats, threshold values of 288 µmol (18 mg l-1) NO3- l-1 and 0.42 µmol (0.04 mg l-1) ortho-PO43- l-1 were defined. Data analysis of the spring water composition indicates that the main source for nutrient and nutrient induced base cation enrichment are nitrate losses from intensively used agricultural fields. The anthropogenically induced but regionally different chemical processes in subsoil and aquifers can result in different levels of nutrient pollution in springs. Further regulations for nitrate and phosphate application are required to conserve and restore groundwater fed ecosystems in Europe.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens. Phylogenetic and in silico expression analyses were performed to identify and characterise DOG1 domain-containing genes in P. patens. Germination assays were performed to characterise a Ppdog1-like1 mutant, and replacement with AtDOG1 was carried out. Yeast two-hybrid assays were used to test the interaction of the PpDOG1-like protein with DELLA proteins from P. patens and A. thaliana. P. patens possesses nine DOG1 domain-containing genes. The DOG1-like protein PpDOG1-L1 (Pp3c3_9650) interacts with PpDELLAa and PpDELLAb and the A. thaliana DELLA protein AtRGA in yeast. Protein truncations revealed the DOG1 domain as necessary and sufficient for interaction with PpDELLA proteins. Spores of Ppdog1-l1 mutant germinate faster than wild type, but replacement with AtDOG1 reverses this effect. Our data demonstrate a role for the PpDOG1-LIKE1 protein in moss spore germination, possibly alongside PpDELLAs. This suggests a conserved DOG1 domain function in germination, albeit with differential adaptation of regulatory networks in seed and spore germination.