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The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type–specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1 , which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth. [ABSTRACT FROM AUTHOR]

RNase H1s are associated with growth and development in both plants and animals, while the roles of RNase H1s in bryophytes have been rarely reported. Our previous data found that PpRNH1A, a member of the RNase H1 family, could regulate the development of Physcomitrium (Physcomitrella) patens by regulating the auxin. In this study, we further investigated the biological functions of PpRNH1A and found PpRNH1A may participate in response to heat stress by affecting the numbers and the mobilization of lipid droplets and regulating the expression of heat-related genes. The expression level of PpRNH1A was induced by heat stress (HS), and we found that the PpRNH1A overexpression plants (A-OE) were more sensitive to HS. At the same time, A-OE plants have a higher number of lipid droplets but with less mobility in cells. Consistent with the HS sensitivity phenotype in A-OE plants, transcriptomic analysis results indicated that PpRNH1A is involved in the regulation of expression of heat-related genes such as DNAJ and DNAJC. Taken together, these results provide novel insight into the functions of RNase H1s. [ABSTRACT FROM AUTHOR]

In multi-step phosphorelay (MSP) signaling, upon reception of various environmental signals, histidine kinases (HKs) induce autophosphorylation and subsequent phosphotransfer to partner histidine-containing phosphotransfer proteins (HPts). Recently, we reported that (i) two Per-Arnt-Sim (PAS) domain-containing HKs (PHK1 and PHK2) of the moss Physcomitrium (Physcomitrella) patens suppressed red light-induced branching of protonema tissue, and (ii) they interacted with partner HPts (HPt1 and HPt2) in the nucleus in the dark while cytoplasmic interactions also occurred under red light. Here we demonstrate that PHK1 is diurnally regulated, i.e. , it is localized and interacts with HPt1 and HPt2 in the nucleus at night whereas these activities reversibly expand and become nucleocytoplasmic in the day. In the dark, PHK1 interacts with HPts only in the nucleus, even in subjective daytime, indicating that endogenous regulation by the circadian clock is not involved. These results suggest that PHK1 is a regulator of moss' adaptation to a light environment on a daily timescale. We discuss a possible regulatory mechanism for the branching of protonema. [Display omitted] • Localization of PHK1 is diurnally regulated, nuclear by night and nucleocytoplasmic by day. • Interactions of PHK1 with HPt1/2 are similarly regulated, nuclear by night and nucleocytoplasmic by day. • These properties of PHK1 provide a novel regulatory framework of plant MSPs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Multi‐step phosphorelay (MSP) is a broadly distributed signaling system in organisms. In MSP, histidine kinases (HKs) receive various environmental signals and transmit them by autophosphorylation followed by phosphotransfer to partner histidine‐containing phosphotransfer proteins (HPts). Previously, we reported that Per‐Arnt‐Sim (PAS) domain‐containing HK1 (PHK1) and PHK2 of the moss Physcomitrium (Physcomitrella) patens repressed red light‐induced protonema branching, a critical step in the moss life cycle. In plants, PHK homolog‐encoding genes are conserved only in early‐diverging lineages such as bryophytes and lycophytes. PHKs‐mediated signaling machineries attract attention especially from an evolutionary viewpoint, but they remain uninvestigated. Here, we studied the P. patens PHKs focusing on their subcellular patterns of localization and interaction with HPts. Yeast two‐hybrid analysis, a localization assay with a green fluorescent protein, and a bimolecular fluorescence complementation analysis together showed that PHKs are localized and interact with partner HPts mostly in the nucleus, as unprecedented features for plant HKs. Additionally, red light triggered the interactions between PHKs and HPts in the cytoplasm, and light co‐repressed the expression of PHK1 and PHK2 as well as genes encoding their partner HPts. Our results emphasize the uniqueness of PHKs‐mediated signaling machineries, and functional implications of this uniqueness are discussed. [ABSTRACT FROM AUTHOR]

Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen. [ABSTRACT FROM AUTHOR]

Summary: ANGUSTIFOLIA (AN) is a plant‐specific subfamily of the CtBP/BARS/AN family, characterized by a plant‐specific C‐terminal domain of approximately 200 amino acids. Previously, we revealed that double knockout (DKO) lines of Physcomitrium (Physcomitrella) patensANGUSTIFOLIA genes (PpAN1‐1 and PpAN1‐2) show defects in gametophore height and the lengths of the seta and foot region of sporophytes, by reduced cell elongation. In addition to two canonical ANs, the genome of P. patens has two atypical ANs without a coding region for a plant‐specific C‐terminus (PpAN2‐1 and PpAN2‐2); these were investigated in this study. Similar to PpAN1s, both promoters of the PpAN2 genes were highly active in the stems of haploid gametophores and in the middle‐to‐basal region of young diploid sporophytes that develop into the seta and foot. Analyses of PpAN2‐1/2‐2 DKO and PpAN quadruple knockout (QKO) lines implied that these four AN genes have partially redundant functions to regulate cell elongation in their expression regions. Transgenic strains harboring P. patens α‐tubulin fused to green fluorescent protein, which were generated from a QKO line, showed that the orientation of the microtubules in the gametophore tips in the PpAN QKO lines was unchanged from the wild‐type and PpAN1‐1/1‐2 DKO plants. In addition to both PpAN2‐1 and PpAN2‐2, short Arabidopsis AN without the C‐terminus of 200 amino acids could rescue the Arabidopsis thaliana an‐1 phenotypes, implying AN activity is dependent on the N‐terminal regions. Significance Statement: ANGUSTIFOLIA (AN) regulates cell expansion in Arabidopsis thaliana and the moss Physcomitrium (Physcomitrella) patens. Analysis of two atypical paralogs of P. patens AN (PpAN2‐1 and PpAN2‐2) indicated that similar to two canonical PpAN1s, both PpAN2s have cell expansion functions in the gametophore stem and sporophyte seta and foot regions, and can recover the an‐1 mutant phenotypes of A. thaliana, although they have no plant‐specific C‐terminal regions of AN. [ABSTRACT FROM AUTHOR]

Similarly to other plant genomes of similar size, more than half of the genome of P. patens is covered by Transposable Elements (TEs). However, the composition and distribution of P. patens TEs is quite peculiar, with Long Terminal Repeat (LTR)-retrotransposons, which form patches of TE-rich regions interleaved with gene-rich regions, accounting for the vast majority of the TE space. We have already shown that RLG1, the most abundant TE in P. patens, is expressed in non-stressed protonema tissue. Here we present a non-targeted analysis of the TE expression based on RNA-Seq data and confirmed by qRT-PCR analyses that shows that, at least four LTR-RTs (RLG1, RLG2, RLC4 and tRLC5) and one DNA transposon (PpTc2) are expressed in P. patens. These TEs are expressed during development or under stresses that P. patens frequently faces, such as dehydratation/rehydratation stresses, suggesting that TEs have ample possibilities to transpose during P. patens life cycle. Indeed, an analysis of the TE polymorphisms among four different P. patens accessions shows that different TE families have recently transposed in this species and have generated genetic variability that may have phenotypic consequences, as a fraction of the TE polymorphisms are within or close to genes. Among the transcribed and mobile TEs, tRLC5 is particularly interesting as it concentrates in a single position per chromosome that could coincide with the centromere, and its expression is specifically induced in young sporophyte, where meiosis takes place.

Similarly to other plant genomes of similar size, more than half of the genome of P. patens is covered by Transposable Elements (TEs). However, the composition and distribution of P. patens TEs is quite peculiar, with Long Terminal Repeat (LTR)-retrotransposons, which form patches of TE-rich regions interleaved with gene-rich regions, accounting for the vast majority of the TE space. We have already shown that RLG1, the most abundant TE in P. patens , is expressed in non-stressed protonema tissue. Here we present a non-targeted analysis of the TE expression based on RNA-Seq data and confirmed by qRT-PCR analyses that shows that, at least four LTR-RTs (RLG1, RLG2, RLC4 and tRLC5) and one DNA transposon (PpTc2) are expressed in P. patens. These TEs are expressed during development or under stresses that P. patens frequently faces, such as dehydratation/rehydratation stresses, suggesting that TEs have ample possibilities to transpose during P. patens life cycle. Indeed, an analysis of the TE polymorphisms among four different P. patens accessions shows that different TE families have recently transposed in this species and have generated genetic variability that may have phenotypic consequences, as a fraction of the TE polymorphisms are within or close to genes. Among the transcribed and mobile TEs, tRLC5 is particularly interesting as it concentrates in a single position per chromosome that could coincide with the centromere, and its expression is specifically induced in young sporophyte, where meiosis takes place. [ABSTRACT FROM AUTHOR]

Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

• Vegetative transcriptomes of Funaria hygrometrica and Physcomitrium pyriforme (Funariaceae, mosses) were compared and characterized against the genome of the closely related model taxon, Physcomitrium (Physcomitrella) patens. • High divergence in gene family size, reflecting significant gains and losses emerges from these comparisons, in sharp contrast with the high vegetative morphological similarity of these mosses. • The observed genomic divergences may support the hypothesis of the diversification of mosses being shaped primarily by metabolic or life history versus morphological, transformations or innovations. A new paradigm has slowly emerged regarding the diversification of bryophytes, with inferences from molecular data highlighting a dynamic evolution of their genome. However, comparative studies of expressed genes among closely related taxa is so far missing. Here we contrast the dimensions of the vegetative transcriptome of Funaria hygrometrica and Physcomitrium pyriforme against the genome of their relative, Physcomitrium (Physcomitrella) patens. These three species of Funariaceae share highly conserved vegetative bodies, and are partially sympatric, growing on mineral soil in mostly temperate regions. We analyzed the vegetative gametophytic transcriptome of F. hygrometrica and P. pyriforme and mapped short reads, transcripts, and proteins to the genome and gene space of P. patens. Only about half of the transcripts of F. hygrometrica map to their ortholog in P. patens , whereas at least 90% of those of P. pyriforme align to loci in P. patens. Such divergence is unexpected given the high morphological similarity of the gametophyte but reflects the estimated times of divergence of F. hygrometrica and P. pyriforme from P. patens , namely 55 and 20 mya, respectively. The newly sampled transcriptomes bear signatures of at least one, rather ancient, whole genome duplication (WGD), which may be shared with one reported for P. patens. The transcriptomes of F. hygrometrica and P. pyriforme reveal significant contractions or expansions of different gene families. While transcriptomes offer only an incomplete estimate of the gene space, the high number of transcripts obtained suggest a significant divergence in gene sequences, and gene number among the three species, indicative of a rather strong, dynamic genome evolution, shaped in part by whole, partial or localized genome duplication. The gene ontology of their specific and rapidly-evolving protein families, suggests that the evolution of the Funariaceae may have been driven by the diversification of metabolic genes that may optimize the adaptations to environmental conditions, a hypothesis well in line with ecological patterns in the genetic diversity and structure in seed plants. [ABSTRACT FROM AUTHOR]

As a bryophyte and model plant, the moss Physcomitrium (Physcomitrella) patens (P. patens) is particularly well adapted to hormone evolution studies. Gene targeting through homologous recombination or CRISPR-Cas9 system, genome sequencing, and numerous transcriptomic datasets has allowed for molecular genetics studies and much progress in Evo-Devo knowledge. As to strigolactones, like for other hormones, both phenotypical and transcriptional responses can be studied, in both WT and mutant plants. However, as in any plant species, medium- to large-scale phenotype characterization is necessary, owing to the general high phenotypic variability. Therefore, many biological replicates are required. This may translate to large amount of the investigated compounds, particularly expensive (or difficult to synthesize) in the case of strigolactones. These issues prompted us to improve existing methods to limit the use of scarce/expensive compounds, as well as to simplify subsequent measures/sampling of P. patens. We hence scaled up well-tried experiments, in order to increment the number of tested genotypes in one given experiment.In this chapter, we will describe three methods we set up to study the response to strigolactones and related compounds in P. patens.