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The genome sequence of Hyocomium armoricum (Brid.) Wijk & Margad. (Hypnales: Hypnaceae).

We present a genome assembly of Hyocomium armoricum (Flagellate Feather-moss; Streptophyta; Bryopsida; Hypnales; Hypnaceae). The genome sequence has a total length of 333.60 megabases. Most of the assembly (99.59%) is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial sequence has a length of 104.43 kilobases and the plastid genome assembly has a length of 123.84 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

Flagellate Feather-moss

Moss BRCA2 lacking the canonical DNA-binding domain promotes homologous recombination and binds to DNA.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2's functional diversity across species.

BRCA2 Protein

Distinct types of short open reading frames are translated in plant cells.

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Bryopsida

New insights into bryophyte arabinogalactan-proteins from a hornwort and a moss model organism.

Two bryophyte models, the hornwort Anthoceros agrestis (Anthoceros) and the moss Physcomitrium patens (Physcomitrium), were analyzed for the presence of arabinogalactan-proteins (AGPs), as the emergence of these signaling glycoproteins in evolution is still under debate. AGPs of both species had a galactan core structure similar to that of other bryophyte and fern AGPs, but different from angiosperm AGPs, as 1,6-linked pyranosidic galactose was almost absent. In the Physcomitrium AGP, furanosidic arabinose (Araf) linkages were mainly terminal (10%) or 5-linked (13%), while in Anthoceros, terminal Araf dominated (26%) and was accompanied by very low amounts of 1,3-Araf and pyranosidic terminal Ara. Unusual 3-O-methylated pyranosidic rhamnose, which has never been detected in cell walls of angiosperms, occurred in both bryophyte AGPs (5% in Anthoceros, 10% in Physcomitrium AGP). This was comparable to AGPs of other spore-producing land plants. A bioinformatic search in the genomes of 14 bryophyte species revealed that most hornworts lack sequences encoding GPI-anchored classical AGPs. Generally, hornworts contained fewer sequences for AGP protein backbones compared with the liverwort Marchantia polymorpha and the moss P. patens. All of them comprise sequences for chimeric AGPs, and among those, surprisingly xylogen-like AGPs. Homologous sequences encoding glycosyltransferases and other enzymes involved in the synthesis and decoration of the AGP galactan framework were present in all bryophyte genomes. Immunocytochemistry of Anthoceros tissue detected AGPs at the plasma membrane/cell wall interface but also at the tonoplast, suggesting new functions of AGPs in bryophytes.

Mucoproteins

Comprehensive identification and analysis of clusters of tandemly duplicated genes reveal their contributions to adaptive evolution of green plants.

Tandem gene duplication occurred more frequently compared with the episodic whole-genome duplication (WGD), providing a continuous supply of genetic material for evolutionary innovation and adaptation to changing environments. The rising roles of clusters of tandemly duplicated genes (CTDGs) in the evolution of phenotypic diversity have been unraveled in mammals. However, the content and biological roles of CTDGs remain largely unknown in plants. Here, we comprehensively identified CTDGs in 220 published plant genomes representing major lineages of green plants. The number of CTDGs showed great variation across taxa, ranging from 0 to 6028. The size of CTDGs varied from 2 to 47 genes, with small clusters containing two members predominating. Interestingly, significant expansion of CTDGs was found in early-diverging land plants and is closely associated with the evolution of key traits (e.g., ABA response, plant cuticle, UV-B resistance) required for plants to conquer terrestrial environments. Functional enrichment analysis revealed conserved and specialized functional profiles among different sizes of CTDGs in both Arabidopsis thaliana and the bryophyte Physcomitrium patens. Small CTDGs were enriched in fundamental stress responses, including protein modification, signal transduction, and responses to diverse stress stimuli, while large CTDGs were enriched in more sophisticated processes such as plant hormone biosynthesis and signaling, plant-microbe interactions, and reproductive processes. Expression pattern analyses of CTDGs under different stress conditions in A. thaliana and P. patens revealed that the highest number of CTDGs showed differential expression under drought stress, suggesting important roles of CTDGs in the evolution of desiccation tolerance in early land plants. The results of this study provide new additions to our knowledge about the abundance of CTDGs across green plants and reveal their important contributions to enable plants to overcome stressful environments on land.

Gene Duplication

Phosphoproteomics analysis provides novel insight into the mechanisms of extreme desiccation tolerance of the desert moss Syntrichia caninervis.

Syntrichia caninervis is a model species for research on desiccation tolerance (DT) because it is capable of rapidly responding to drastic changes in water conditions. Phosphorylation, a key post-translational modification process that is rapid and reversible, enables the rapid regulation of protein functions, aiding plants to quickly adapt to changing environments. Modifications to phosphorylation may play a crucial role in the DT of S. caninervis, although no studies have been published. Here, we report a 4D label-free high-resolution dynamic proteomic and phosphoproteomic analysis of S. caninervis during dehydration and rehydration, allowing for the quantification of 2854 proteins and 1177 phosphoproteins, including 1447 differentially expressed proteins (DEPs) and 699 differentially phosphorylated proteins (DPPs). Among the phosphoproteins, 36.5% displayed changes in protein abundance. The proteomic and phosphoproteomic changes involved proteins (DEPs and DPPs) that were mainly involved in photosynthesis, glutathione metabolism, the citrate cycle, and the biosynthesis of secondary metabolism pathways during dehydration. During rehydration, DEPs and DPPs were mainly associated with processes related to ribosome and energy metabolism. In summary, during dehydration, phosphorylation mainly regulates signal transduction and metabolic processes, allowing plants to adapt to a loss of water. During rehydration, phosphorylation controls repair and recovery mechanisms, restoring metabolic activity and reestablishing cellular functions. ScDHAR1, a protein involved in glutathione metabolism, was differentially phosphorylated at two serine sites (S29 and S218) in response to desiccation. Further analysis revealed that phosphorylation of S29/S218 in ScDHAR1 significantly increased its enzymatic activity, thereby enhancing the DT of S. caninervis in situ. This work establishes a phosphoprotein database for a DT moss. These findings not only broaden our understanding of S. caninervis DT but also fill knowledge gaps in the field of phosphoproteomics in DT mosses, while providing valuable data resources for future related research.

Phosphoproteins

STEMIN transcription factor drives selective chromatin remodeling for gene activation within a relaxed chromatin during reprogramming in the moss Physcomitrium patens.

Land plants exhibit remarkable cellular plasticity, readily reprogramming differentiated cells into stem cells in response to internal and external stimuli. While chromatin remodeling is crucial for cellular reprogramming, its interplay with gene expression during reprogramming into stem cells remains elusive. In the moss Physcomitrium patens, wounding induces reprogramming of leaf cells facing wounded cells to change into chloronema apical stem cells through the activation of the AP2/ERF transcription factor STEMIN. In this study, we employed multimodal single-nuclei RNA and ATAC sequencing to explore the interplay between gene expression and chromatin dynamics during STEMIN-mediated reprogramming. Profiling 20&#x2009;883 single-nuclei from gametophores, protonemata, and cut leaves, we identified 11 distinct cell types including reprogramming leaf cells. Our analysis revealed that reprogramming leaf cells exhibit a partly relaxed chromatin landscape and STEMIN transcription factors selectively enhance accessibility at specific genomic loci essential for stem cell formation. Thus, our results indicate that wounding initiates a broad chromatin relaxation, creating a permissive environment and specific transcription factors act to refine this permissive state by specifically relaxing chromatin regions critical for reprogramming.

Bryopsida

TRB proteins in moss reveal their evolutionarily conserved roles in plant development and telomere maintenance.

Telomere repeat binding (TRB) proteins are plant-specific proteins with a unique domain structure distinct from telomerebinding proteins in animals and yeast. While extensively studied in seed plants, their role in early-diverging plant lineages remains largely unexplored. Here, we investigate TRB proteins in a model moss, Physcomitrium patens, to assess their evolutionary conservation and functional significance. Functional analysis using single knockout mutants revealed that individual PpTRB genes are essential for normal development, with mutants exhibiting defects in the two-dimensional (protonemal) stage, and more prominently, in the formation of three-dimensional (gametophore) structures. Some double mutants displayed telomere shortening, a phenotype also observed in TRB-deficient seed plants, indicating a conserved role for TRBs in telomere maintenance. Transcriptome profiling of TRB mutants revealed altered expression of genes associated with transcriptional regulation and stimulus response in protonema. Subcellular localization studies across various plant cell types confirmed that PpTRBs, like their seed plant counterparts, localize prevalently to the plant nucleus and mutually interact. In bryophytes, TRBs form a monophyletic group that mirrors the species phylogeny, whereas in seed plants, TRBs have diversified into two distinct monophyletic groups. Our findings provide the first comprehensive characterization of TRB proteins in non-vascular plants and demonstrate their conserved roles in telomere maintenance, with additional implications for plant development and gene regulation across land plant lineages.

Bryopsida

MAdLandExpression: integrating sexual reproduction into the Physcomitrium patens expression atlas.

Physcomitrium patens is a bryophyte model system particularly valuable for evolutionary developmental and comparative genomics studies. Sexual reproduction in bryophytes offers unique insights into the evolution of land plant reproduction. Unlike seed plants, bryophytes have a dominant gametophyte phase and provide significant advantages for studying sexual reproduction, such as the possibility to maintain embryo-lethal mutants through vegetative propagation or the presence of motile male gametes. More than 25&#x2009;years after the first publications of transcriptomic data for P. patens, expression data of most developmental stages of P. patens as well as its responses to various biotic and abiotic perturbations have been represented by microarrays or RNA-seq datasets. To facilitate the use of such data, we introduce the MAdLandExpression atlas as a successor of PEATmoss (Physcomitrium Expression Atlas Tool), integrating its 109 P. patens expression experiments and expanding it with 20 recently published RNA-seq samples of sexual reproduction stages, thus completing the coverage of the P. patens life cycle. The MAdLandExpression atlas also introduces new features for data visualization and analysis, such as the comparison of samples from multiple datasets and gene set normalization. Using this tool, the sexual reproduction dataset was analyzed, identifying genes potentially important for egg and sperm cell development, and confirming the behavior of known key genes in sexual development observed in previous studies.

Bryopsida

Insights into the regulatory roles of LIKE-HETEROCHROMATIN PROTEIN 1 and its targeting to different nuclear compartments modulated by NLS and the conserved domains in the moss Physcomitrium patens.

LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) is a polycomb group protein that exists in shared multiprotein complexes that harbor core PRC1 and PRC2 proteins. We previously characterized LHP1 in the moss Physcomitrium patens and showed that its function is closely linked with regulation of RNA metabolic processes and the protein is distributed in the nucleoplasm, subnuclear foci, and the nucleolus. To gain mechanistic insight into PpLHP1-mediated gene regulation, in the present study genome-wide changes in transcript profiles of genes affected by loss-of-PpLHP1 function were studied using pplhp1 mutants. RNA-seq analysis reveals a key role for PpLHP1 in regulating energy metabolic processes, ribosome-related pathways, stress signaling/responsive pathways, DNA transcription, etc. ChIP using H3K27me3 coupled with qRT-PCR shows that PpLHP1 suppresses transcription at 5S rRNA promoters and the untimely activation of genes regulating developmental transition by PRC2-dependent and independent mechanisms. To study how PpLHP1 finds its targets in different nuclear compartments and the roles of the multiple NLSs and the conserved domains in guiding the protein, FRAP and deletion studies were performed. These show that PpLHP1 is a mobile protein that diffuses freely in the nucleoplasmic space showing different retention times in the nucleolus, nucleoplasm, and the subnuclear foci indicating its differential affinity for targets at these sites. Expression of PpLHP1 fragments in protonema cells and its subsequent visualization under confocal microscope shows that localization of PpLHP1 to different subnuclear compartments is guided by the monopartite NLS2, CD, and CSD that also play a key role in promoting subnuclear foci formation in the nucleoplasm.

Bryopsida