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The evolution of green plants from aquatic to terrestrial environments is thought to have been facilitated by the acquisition of gametangia, specialized multicellular organs housing gametes. Antheridia and archegonia, responsible for producing and protecting sperm and egg cells, undergo formative cell divisions to produce a cell to differentiate into germ cell lineages and the other cell to give rise to surrounding structures. However, the genes governing this process remain unidentified. We isolated genes expressed during gametangia development from previously established gene-trap lines of Physcomitrium patens and characterized their function during gametangia formation. We identified P. patens LATERAL SUPPRESSOR 1 (PpLAS1) from the gene-trap library, encoding a GRAS transcription factor. The double-deletion mutant with its paralog PpLAS2 failed to form inner cells in both gametangia. PpLASs are expressed in cells undergoing formative cell division, and introducing PpLAS1 into the double-deletion mutant successfully rescued the phenotype. These findings underscore the pivotal role of PpLASs in regulating formative cell divisions, ensuring the separation of reproductive cell lineages from surrounding cells in antheridia and archegonia. Furthermore, they suggest a link between PpLASs and the evolutionary origin of male and female gametangia in the common ancestor of land plants., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Cell survival depends on the maintenance of cell homeostasis that involves all the biochemical, genomic and transport processes that take place in all the organelles within a eukaryote cell. In particular, ion homeostasis is required to regulate the membrane potential and solute transport across all membranes, any alteration in these parameters will reflect in the malfunctioning of any organelle, and consequently, in the development of the organism. In plant cells, sodium transporters play a central role in keeping the concentrations of this cation across all membranes under physiological conditions to prevent its toxic effects. HKT transporters are a family of membrane proteins exclusively present in plants, with some homologs present in prokaryotes. HKT transporters have been associated to salt tolerance in plants, retrieving any leak of the cation into the xylem, or removing it from aerial parts, including the flowers, to be transported to the roots along the phloem. This function has been assigned as most of the HKT transporters are located at the plasma membrane. Here, we report the localization of the HKT from Physcomitrium patens to the thylakoid membrane, reminiscent of the prokaryote origin of these family of transporters. Mutation of PpHKT leads to several alterations in the phenotype of the organism, including the lack of sporophyte formation, and changes in expression of many genes. These alterations suggest that the breakdown in chloroplast ion homeostasis triggers a signalling cascade to the nucleus to communicate its status, being important for the moss to complete its life cycle., (© 2025 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cell plate formation in plants is a complex process orchestrated by the targeted delivery of Golgi-derived and endosomal vesicles containing cell plate components to the phragmoplast midzone. It has long been hypothesized that vesicles are directionally transported along phragmoplast microtubules by motor proteins. However, the mechanisms governing the accumulation and immobilization of vesicles at the phragmoplast midzone remain elusive, and the motor protein responsible has yet to be identified. Here we show that the plant-specific class II kinesin-12 (kinesin12-II) functions as a motor protein that drives vesicle transport towards the phragmoplast midzone in the moss Physcomitrium patens. In the kinesin12-II mutant, the directional movement of cell plate materials towards the midzone and their retention were abolished, resulting in delayed cell plate formation and phragmoplast disassembly. A macroscopic phenotype arising from kinesin12-II disruption was the impediment to gametophore development. We showed that this defect was attributable to the production of aneuploid and polyploid cells in the early gametophore, where chromosome missegregation and cytokinesis failure occurred. These findings suggest that plant kinesin-12 has evolved to acquire a unique and critical function that facilitates cell plate formation in the presence of phragmoplasts., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

The sexual reproductive organs of bryophytes - in which gametes necessary for fertilization are produced, namely, male antheridia and female archegonia - are formed from vegetative haploid gametophytes. In dioicous bryophytes such as Marchantia polymorpha, the genes within the sex-determining regions in distinct sexual strains have been identified. However, in monoicous bryophytes such as Physcomitrium patens, how the two sex fates are specified on the same gametophyte remained unknown. Here, we identified an RWP-RK domain-containing transcription factor in P. patens, PpRKD, as a factor required for the development of female organs, based on the absence of archegonia in loss-of-function Pprkd mutants and the specific expression of PpRKD in archegonia. When ectopically induced, the expression of PpRKD resulted in the repression of antheridial development and the emergence of archegonium-like organs. Furthermore, the young primordia inside the antheridial bundle displayed typical archegonial division patterns, suggesting that PpRKD confer female fate to antheridium primordia. This study represents the first instance where the function of sex determination has been identified among RKD orthologs in land plants. This finding should provide a new framework for the molecular evolutionary context of the genes in the RKD family, considering the recent elucidation of their roles in algae., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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 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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jessica M. Budke reports financial support was provided by US National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Studying morphological novelties offers special insights into developmental biology and evolution. The inflated calyx syndrome (ICS) is a largely unrecognized but fascinating feature of flower development, where sepals form balloon-like husks that encapsulate fruits. Despite its independent emergence in many lineages of flowering plants, the genetic and molecular mechanisms of ICS remain unknown. Early studies in the Solanaceae genus Physalis put forth key roles of MADS-box genes in ICS. However, recent work suggests these classical floral identity transcription factors were false leads. With newfound capabilities that allow rapid development of genetic systems through genomics and genome editing, Physalis has re-emerged as the most tractable model species for dissecting ICS. This review revisits current understanding of ICS and highlights how recent advancements enable a reset in the search for genetic and molecular mechanisms using unbiased, systematic approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plants rely on solar energy to synthesize ATP and NADPH for photosynthetic carbon fixation and all cellular need. Mitochondrial respiration is essential in plants, but this may be due to heterotrophic bottlenecks during plant development or because it is also necessary in photosynthetically active cells. In this study, we examined in vivo changes of cytosolic ATP concentration in response to light, employing a biosensing strategy in the moss Physcomitrium patens and revealing increased cytosolic ATP concentration caused by photosynthetic activity. Plants depleted of respiratory Complex I showed decreased cytosolic ATP accumulation, highlighting a critical role of mitochondrial respiration in light-dependent ATP supply of the cytosol. Consistently, targeting mitochondrial ATP production directly, through the construction of mutants deficient in mitochondrial ATPase (complex V), led to drastic growth reduction, despite only minor alterations in photosynthetic electron transport activity. Since P. patens is photoautotrophic throughout its development, we conclude that heterotrophic bottlenecks cannot account for the indispensable role of mitochondrial respiration in plants. Instead, our results support that mitochondrial respiration is essential for ATP provision to the cytosol in photosynthesizing cells. Mitochondrial respiration provides metabolic integration, ensuring supply of cytosolic ATP essential for supporting plant growth and development., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species-the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis-consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The hyperaccumulation potential of zinc (Zn) and cadmium (Cd) and their synergistic effects were examined in relation to Christmas moss (Vesicularia montagnei (Bél) Broth., Hypnaceae), an aquatic and terrestrial moss, dosed with Cd (Cd1 and Cd2), Zn (Zn1 and Zn2) and combined Zn and Cd (Cd1Zn1 and Cd2Zn2). Zinc promoted plant growth and development, particularly in the highest Zn and combined Zn/Cd treatments (Zn2 and Cd2Zn2). The Zn treatment resulted in substantial moss chlorophyll content and highest percentage relative growth rate in biomass value (0.23 mg L -1 and 106.8%, respectively); however, the Cd2Zn2 treatment achieved maximal production of chlorophyll a and total chlorophyll (0.29 and 0.51 mg L -1 , respectively) due to synergistic effects. These findings suggest that Christmas moss is a highly metal-tolerant and adaptable bryophyte species. Zinc was essential for reducing the detrimental effects of Cd while simultaneously promoting moss growth and biomass development. Furthermore, Christmas moss exhibited hyperaccumulation potential for Cd and Zn in the Cd2Zn2 and Zn alone treatments, as evidenced by highest Cd and Zn values in gametophores (1002 and 18,596 mg per colony volume, respectively). Using energy dispersive X-ray fluorescence (EDXRF) spectrometry, atomic percentages of element concentrations in moss gametophores in the Zn2, Cd2 and combined Zn/Cd treatments were generally in the order: K > Ca > P > Zn > Cd. When comparing the atomic percentages of Zn and Cd in gametophores, it is likely that the higher atomic percentage of Zn was because this element is essential for plant growth and development., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

This study investigated the responses of Didymodon constrictus and Hypnum plumaeforme to different light qualities emitted by light-emitting diodes (LEDs), including white light (WL), red light (RL), blue light (BL), yellow light (YL), green light (GL), and a combination of red and blue light (R1B1L). The research analyzed the fluorescence imaging, photosynthetic pigments, coloration, and growth characteristics related to antioxidant enzymes in these two moss species. The results indicated that R1B1L significantly enhanced the content of photosynthetic pigments, maximum relative electron transport rate (rETRmax), saturation light intensity (IK), and the greenness of the moss. RL improved the maximum quantum yield (Fv/Fm), the light energy efficiency of H. plumaeforme and effective quantum yield in both moss species. In contrast, BL notably increased non-photochemical quenching (NPQ), photochemical quenching (qp), and the steady-state fluorescence decrease ratio (RFD) in H. plumaeforme. The application of GL significantly increases the maximum photon yield (Fv/Fm) in D. constrictus, as well as the light energy efficiency and elongation length, resulting in a shift in the color composition of both moss species towards yellow. Among the light treatments, R1B1L had the highest induction rate and promotional effect on the growth of both moss species. These mosses absorbed GL and RL effectively, while BL played a crucial role in the dissipation of heat and electron transfer in H. plumaeforme. This research provides valuable insights for the regulation of LED light environments and the physiological adaptability of moss in artificial cultivation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiurong Wang reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2021 John Wiley & Sons Ltd.)

R-loops, consisting of a DNA:RNA hybrid and a single-stranded DNA (ssDNA), form naturally as functional chromosome structures and are crucial in many vital biological processes. However, disrupted R-loop homeostasis will threat to the integrity and stability of genome. As the endonuclease, RNase H1 can efficiently recognize and remove excess R-loops to protect organisms from DNA damage induced by R-loop over-accumulation. Here, we investigated the function of RNase H1 in Physcomitrium (Physcomitrella) patens to illustrate its important role in the evolution of plants. We found that PpRNH1A dysfunction seriously affected shoot growth and branch formation in P. patens, revealing a noticeable functional difference between PpRNH1A and AtRNH1A of Arabidopsis. Furthermore, auxin signaling was significantly affected at the transcriptional level in PpRNH1A mutant plants, as a result of the accumulation of R-loops at several auxin-related genes. This study provides evidence that PpRNH1A regulates the development of P. patens by controlling R-loop formation at specific loci to modulate the transcription of auxin-related genes. It also highlights the interspecific functional differences between early land plants and vascular plants, despite crucial and conserved role of RNase H1 played in maintaining R-loop homeostasis., (Copyright © 2021. Published by Elsevier B.V.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: Moss PPR-SMR protein PpPPR&#95;64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR&#95;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&#95;64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR&#95;64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR&#95;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&#95;64 KO mutants. This suggests that PpPPR&#95;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&#95;64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens., (© 2020. Springer Nature B.V.)

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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

As an adaptive innovation in plant terrestrialization, cuticle covers the plant surface and greatly contributes to the development and stress tolerance in land plants. Although past decades have seen great progress in understanding the molecular mechanism of cuticle biosynthesis in flowering plants with the contribution of cuticle biosynthesis mutants and advanced cuticle composition profiling techniques, origins and evolution of cuticle biosynthesis are poorly understood. Recent chemical, phylogenomic, and molecular genetic studies on cuticle biosynthesis in early-diverging extant land plant lineages, the bryophytes, shed novel light on the origins and evolution of plant cuticle biosynthesis. In this mini-review, we highlighted these recent advances in the molecular biology of cuticle biosynthesis in bryophytes, and provided evolutionary insights into plant cuticle biosynthesis.

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana Pp PIEZO1 and Pp PIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both Pp PIEZO1 and Pp PIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying β-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between β-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The moss Physcomitrium (Physcomitrella) patens performs efficient homologous recombination in both the nucleus and plastid enabling the study of individual gene function by generating precise inactivation or modification of genes. Polyethylene glycol (PEG)-mediated transformation of protoplasts is routinely used to study the nuclear gene function of P. patens. PEG-mediated protoplast transformation is also applied for plastid transformation of this moss. The efficiency of plastid transformation is quite reliable and one or two homoplasmic transplastomic lines are obtained in a plastid transformation experiment (5 × 10 5 protoplasts) by selection for spectinomycin resistance.

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.

Plants synthesize gibberellin (GA), a diterpenoid hormone, via ent-kaurenoic acid (KA) oxidation. GA has not been detected in the moss Physcomitrium patens despite its ability to synthesize KA. It was recently shown that a KA metabolite, 3OH-KA, was identified as an active regulator of protonema differentiation in P. patens. An inactive KA metabolite, 2OH-KA, was also identified in the moss, as was KA2ox, which is responsible for converting KA to 2OH-KA. In this review, we mainly discuss the GA biosynthetic gene homologs identified and characterized in bryophytes. We show the similarities and differences between the OH-KA control of moss and GA control of flowering plants. We also discuss using recent genomic studies; mosses do not contain KAO, even though other bryophytes do. This absence of KAO in mosses corresponds to the presence of KA2ox, which is absent in other vascular plants. Thus, given that 2OH-KA and 3OH-KA were isolated from ferns and flowering plants, respectively, vascular plants may have evolved from ancestral bryophytes that originally produced 3OH-KA and GA., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2020 American Society of Plant Biologists. All rights reserved.)

The concentrations of trace elements in feather moss Pleurozium schreberi (Brid.) Mitt. were used to indicate the relative levels of air pollution by trace elements in Polish national parks. Pleurozium schreberi was collected from nine national parks. The highest concentrations were recorded in the moss samples from the southern and most industrialised part of the country; the lowest from northern and north-eastern Poland. A comparison of data obtained from Polish national parks in the 1970s and 1990s showed a significant decrease in the concentrations of heavy metals. In the linear covariability estimation, the t quantile approach was used for multi-element comparison. A number of positive covariabilities were observed. This is a result of anthropogenic activity and the geochemical characteristics of the local environment, including crust composition to which soil composition is related. The statistical approach of t quantile to study common relationships between element concentrations can be used in the interpretation of biomonitoring research results in similar studies.

Background: Autophagy is an evolutionarily conserved system for the degradation of intracellular components in eukaryotic organisms. Autophagy plays essential roles in preventing premature senescence and extending the longevity of vascular plants. However, the mechanisms and physiological roles of autophagy in preventing senescence in basal land plants are still obscure., Results: Here, we investigated the functional roles of the autophagy-related gene PpATG3 from Physcomitrella patens and demonstrated that its deletion prevents autophagy. In addition, Ppatg3 mutant showed premature gametophore senescence and reduced protonema formation compared to wild-type (WT) plants under normal growth conditions. The abundance of nitrogen (N) but not carbon (C) differed significantly between Ppatg3 mutant and WT plants, as did relative fatty acid levels. In vivo protein localization indicated that PpATG3 localizes to the cytoplasm, and in vitro Y2H assays confirmed that PpATG3 interacts with PpATG7 and PpATG12. Plastoglobuli (PGs) accumulated in Ppatg3, indicating that the process that degrades damaged chloroplasts in senescent gametophore cells was impaired in this mutant. RNA-Seq uncovered a detailed, comprehensive set of regulatory pathways that were affected by the autophagy mutation., Conclusions: The autophagy-related gene PpATG3 is essential for autophagosome formation in P. patens. Our findings provide evidence that autophagy functions in N utilization, fatty acid metabolism and damaged chloroplast degradation under non-stress conditions. We identified differentially expressed genes in Ppatg3 involved in numerous biosynthetic and metabolic pathways, such as chlorophyll biosynthesis, lipid metabolism, reactive oxygen species removal and the recycling of unnecessary proteins that might have led to the premature senescence of this mutant due to defective autophagy. Our study provides new insights into the role of autophagy in preventing senescence to increase longevity in basal land plants.

DNA damage can result from intrinsic cellular processes and from exposure to stressful environments. Such DNA damage generally threatens genome integrity and cell viability 1 . However, here we report that the transient induction of DNA strand breaks (single-strand breaks, double-strand breaks or both) in the moss Physcomitrella patens can trigger the reprogramming of differentiated leaf cells into stem cells without cell death. After intact leafy shoots (gametophores) were exposed to zeocin, an inducer of DNA strand breaks, the STEM CELL-INDUCING FACTOR 1 (STEMIN1) 2 promoter was activated in some leaf cells. These cells subsequently initiated tip growth and underwent asymmetric cell divisions to form chloronema apical stem cells, which are in an earlier phase of the life cycle than leaf cells and have the ability to form new gametophores. This DNA-strand-break-induced reprogramming required the DNA damage sensor ATR kinase, but not ATM kinase, together with STEMIN1 and closely related proteins. ATR was also indispensable for the induction of STEMIN1 by DNA strand breaks. Our findings indicate that DNA strand breaks, which are usually considered to pose a severe threat to cells, trigger cellular reprogramming towards stem cells via the activity of ATR and STEMINs.

Branching morphogenesis is a widely used mechanism for development [1, 2]. In plants, it is initiated by the emergence of a new growth axis, which is of particular importance for plants to explore space and access resources [1]. Branches can emerge either from a single cell or from a group of cells [3-5]. In both cases, the mother cells that initiate branching must undergo dynamic morphological changes and/or adopt oriented asymmetric cell divisions (ACDs) to establish the new growth direction. However, the underlying mechanisms are not fully understood. Here, using the bryophyte moss Physcomitrella patens as a model, we show that side-branch formation in P. patens protonemata requires coordinated polarized cell expansion, directional nuclear migration, and orientated ACD. By combining pharmacological experiments, long-term time-lapse imaging, and genetic analyses, we demonstrate that Rho of plants (ROP) GTPases and actin are essential for cell polarization and local cell expansion (bulging). The growing bulge acts as a prerequisite signal to guide long-distance microtubule (MT)-dependent nuclear migration, which determines the asymmetric positioning of the division plane. MTs play an essential role in nuclear migration but are less involved in bulge formation. Hence, cell polarity and cytoskeletal elements act cooperatively to modulate cell morphology and nuclear positioning during branch initiation. We propose that polarity-triggered nuclear positioning and ACD comprise a fundamental mechanism for increasing multicellularity and tissue complexity during plant morphogenesis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Main Conclusion: β-carbonic anhydrases, which function in regulating plant growth, C/N status, and stomata number, showed functional redundancy and divergence in Physcomitrella patens. Carbonic anhydrases (CAs) catalyze the interconversion of CO 2 and HCO 3 - . Plants have three evolutionarily unrelated CA families: α-, β-, and γ-CAs. βCAs are abundant in plants and are involved in CO 2 assimilation, stress responses, and stomata formation. Recent studies of βCAs have mainly examined C 3 or C 4 plants, whereas their functions in non-vascular plants are mostly unknown. In this study, phylogenetic analysis revealed that the evolution of βCAs were conserved between subaerial green algae and bryophytes after terrestrialization event, and βCAs from some cyanobacteria might begin evolving for the adaptation of terrestrial environment/habitat. In addition, we investigated the physiological roles of βCAs in the basal land plant Physcomitrella patens. High PpβCA expression levels in different tissues suggest that PpβCAs play important roles in development in P. patens. Plants treated with 1-10 mM NaHCO 3 had higher fresh and dry weight, PpβCA expression, total CA activity, and photosynthetic yield (Fv/Fm) compared with water-treated plants. However, treatment with 10 mM NaHCO 3 influenced the C/N status. Further study of six Ppβca single-gene mutants revealed that PpβCAs have functional redundancy and divergence in regulating the C/N ratio of plants and stomatal formation. This study provides new insight into the physiological roles of βCAs in basal land plants.

Coordinated by ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR), two highly conserved kinases, DNA damage repair ensures genome integrity and survival in all organisms. The Arabidopsis thaliana ( A. thaliana ) orthologues are well characterized and exhibit typical mammalian characteristics. We mutated the Physcomitrella patens ( P. patens ) PpATM and PpATR genes by deleting functionally important domains using gene targeting. Both mutants showed growth abnormalities, indicating that these genes, particularly PpATR , are important for normal vegetative development. ATR was also required for repair of both direct and replication-coupled double-strand breaks (DSBs) and dominated the transcriptional response to direct DSBs, whereas ATM was far less important, as shown by assays assessing resistance to DSB induction and SuperSAGE-based transcriptomics focused on DNA damage repair genes. These characteristics differed significantly from the A. thaliana genes but resembled those in yeast ( Saccharomyces cerevisiae ). PpATR was not important for gene targeting, pointing to differences in the regulation of gene targeting and direct DSB repair. Our analysis suggests that ATM and ATR functions can be substantially diverged between plants. The differences in ATM and ATR reflect the differences in DSB repair pathway choices between A. thaliana and P. patens , suggesting that they represent adaptations to different demands for the maintenance of genome stability.

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 P patens 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

eIF4A is a RNA-stimulated ATPase and helicase. Besides its key role in regulating cap-dependent translation initiation in eukaryotes, it also performs specific functions in regulating cell cycle progression, plant growth and abiotic stress tolerance. Flowering plants encode three eIF4A paralogues, eIF4A1, eIF4A2 and eIF4A3 that share conserved sequence motifs but differ in functions. To date, however, no information is available on eIF4A in basal land plants. In this study we report that genome of the moss Physcomitrella patens encodes multiple eIF4A genes. The encoded proteins possess the highly conserved motifs characteristic of the DEAD box helicases. Spatial expression analysis shows these genes to be ubiquitously expressed in all tissue types with Pp3c6&#95;1080V3.1 showing high expression in filamentous protonemata. Targeted deletion of conserved core motifs in Pp3c6&#95;1080V3.1 slowed protonemata growth and resulted in dwarfing of leafy gametophores suggesting a role for Pp3c6&#95;1080V3.1 in regulating cell division/elongation. Rapid and strong induction of Pp3c6&#95;1080V3.1 under salt stress and slow recovery of knockout plants upon exposure to high salt further suggest Pp3c6&#95;1080V3.1 to be involved in stress management in P. patens. Protein-protein interaction studies that show Pp3c6&#95;1080V3.1 to interact with the Physcomitrella heterogenous ribonucleoprotein, LIF2L1, a transcriptional regulator of stress-responsive genes in Arabidopsis. The results presented in this study provide insight into evolutionary conserved functions of eIF4A and shed light on the novel link between eIF4A activities and stress mitigation pathways/RNA metabolic processes in P. patens.

Key Message: PpCKX1 localizes to vacuoles and is dominantly expressed in the stem cells. PpCKX1 regulates developmental changes with increased growth of the rhizoid and enhances dehydration and salt tolerance. Cytokinins (CKs) are plant hormones that regulate plant development as well as many physiological processes, such as cell division, leaf senescence, control of shoot/root ratio, and reproductive competence. Cytokinin oxidases/dehydrogenases (CKXs) control CK concentrations by degradation, and thereby influence plant growth and development. In the moss Physcomitrella patens, an evolutionarily early divergent plant, we identified six putative CKXs that, by phylogenetic analysis, form a monophyletic clade. We also observed that ProPpCKX1:GUS is expressed specifically in the stem cells and surrounding cells and that CKX1 localizes to vacuoles, as indicated by Pro35S:PpCKX1-smGFP. Under normal growth conditions, overexpression of PpCKX1 caused many phenotypic changes at different developmental stages, and we suspected that increased growth of the rhizoid could affect those changes. In addition, we present evidence that the PpCKX1-overexpressor plants show enhanced dehydration and salt stress tolerance. Taken together, we suggest that PpCKX1 plays regulatory roles in development and adaptation to abiotic stresses in this evolutionarily early land plant species.

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., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The strategies and experimental approaches that led the author to demonstrate the role of auxin in caulonema differentiation in the protonema of the moss Funaria hygrometrica are discussed. In stationary suspension cultures, the status of cell differentiation is regulated by inoculum cell density and auxin level. At low inoculum cell densities, 2-5 µM indole acetic acid (IAA) led to the differentiation of 65-70% caulonema filaments in 5-6 days. Caulonema can also differentiate in auxin-free medium if buffered at pH 5.0 after a lag of 6±1 days. The duration of lag can be manipulated and the cells are capable of responding to auxin at a higher level (3-10 µM) and produce about 20% caulonema after 3 days. This responsiveness or sensitivity to auxin can be enhanced further by growing cells in a nutrient-limited medium buffered at pH 5.0. In this medium, addition of 3 µM IAA led to the differentiation of 75-80% caulonema and rhizoids within 3 to 4 days. Work done in other laboratories has shown that auxin promotes caulonema differentiation in the moss Physcomitrella patens by positively regulating two basic helix-loop-helix type of transcription factor genes namely root hair defective six-like1 (PpRSL1) and PpRSL2 (Jang and Dolan 2011, New Phytologist 192: 319-327).

Background: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata., Methods: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test., Results: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters., Conclusions: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

The radiation of life on Earth was accompanied by the diversification of multicellular body plans in the eukaryotic kingdoms Animalia, Plantae, Fungi and Chromista. Branching forms are ubiquitous in nature and evolved repeatedly in the above lineages. The developmental and genetic basis of branch formation is well studied in the three-dimensional shoot and root systems of land plants, and in animal organs such as the lung, kidney, mammary gland, vasculature, etc. Notably, recent thought-provoking studies combining experimental analysis and computational modeling of branching patterns in whole animal organs have identified global patterning rules and proposed unifying principles of branching morphogenesis. Filamentous branching forms represent one of the simplest expressions of the multicellular body plan and constitute a key step in the evolution of morphological complexity. Similarities between simple and complex branching forms distantly related in evolution are compelling, raising the question whether shared mechanisms underlie their development. Here, we focus on filamentous branching organisms that represent major study models from three distinct eukaryotic kingdoms, including the moss Physcomitrella patens (Plantae), the brown alga Ectocarpus sp. (Chromista), and the ascomycetes Neurospora crassa and Aspergillus nidulans (Fungi), and bring to light developmental regulatory mechanisms and design principles common to these lineages. Throughout the review we explore how the regulatory mechanisms of branching morphogenesis identified in other models, and in particular animal organs, may inform our thinking on filamentous systems and thereby advance our understanding of the diverse strategies deployed across the eukaryotic tree of life to evolve similar forms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.