Genome sequence and cell biological toolbox of the highly regenerative, coenocytic green feather alga Bryopsis.

Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (>100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14 034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other ulvophyceans, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae.

Morphological Innovation Drives Sperm Release in Bryophytes.

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

Physcomitrium patens: A Single Model to Study Oriented Cell Divisions in 1D to 3D Patterning.

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.

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism.

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.

Fundamental mechanisms of the stem cell regulation in land plants: lesson from shoot apical cells in bryophytes.

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.

Quantitative proteomic analysis to capture the role of heat-accumulated proteins in moss plant acquired thermotolerance.

At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defences that can keep heat-labile membranes and proteins functional. A gradual morning pre-exposure to increasing sub-damaging temperatures induces heat-shock proteins (HSPs) that are central to the onset of plant acquired thermotolerance (AT). To gain knowledge on the mechanisms of AT in the model land plant Physcomitrium patens, we used label-free LC-MS/MS proteomics to quantify the accumulated and depleted proteins before and following a mild heat-priming treatment. High protein crowding is thought to promote protein aggregation, whereas molecular chaperones prevent and actively revert aggregation. Yet, we found that heat priming (HP) did not accumulate HSP chaperones in chloroplasts, although protein crowding was six times higher than in the cytosol. In contrast, several HSP20s strongly accumulated in the cytosol, yet contributing merely 4% of the net mass increase of heat-accumulated proteins. This is in poor concordance with their presumed role at preventing the aggregation of heat-labile proteins. The data suggests that under mild HP unlikely to affect protein stability. Accumulating HSP20s leading to AT, regulate the activity of rare and specific signalling proteins, thereby preventing cell death under noxious heat stress.

High levels of glucose alter Physcomitrella patens metabolism and trigger a differential proteomic response.

Sugars act not only as substrates for plant metabolism, but also have a pivotal role in signaling pathways. Glucose signaling has been widely studied in the vascular plant Arabidopsis thaliana, but it has remained unexplored in non-vascular species such as Physcomitrella patens. To investigate P. patens response to high glucose treatment, we explored the dynamic changes in metabolism and protein population by applying a metabolomic fingerprint analysis (DIESI-MS), carbohydrate and chlorophyll quantification, Fv/Fm determination and label-free untargeted proteomics. Glucose feeding causes specific changes in P. patens metabolomic fingerprint, carbohydrate contents and protein accumulation, which is clearly different from those of osmotically induced responses. The maximal rate of PSII was not affected although chlorophyll decreased in both treatments. The biological process, cellular component, and molecular function gene ontology (GO) classifications of the differentially expressed proteins indicate the translation process is the most represented category in response to glucose, followed by photosynthesis, cellular response to oxidative stress and protein refolding. Importantly, although several proteins have high fold changes, these proteins have no predicted identity. The most significant discovery of our study at the proteome level is that high glucose increase abundance of proteins related to the translation process, which was not previously evidenced in non-vascular plants, indicating that regulation by glucose at the translational level is a partially conserved response in both plant lineages. To our knowledge, this is the first time that metabolome fingerprint and proteomic analyses are performed after a high sugar treatment in non-vascular plants. These findings unravel evolutionarily shared and differential responses between vascular and non-vascular plants.

A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for This GTPase in Subcellular and Tissue-Level Patterning.

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.

Rho of Plants GTPases and Cytoskeletal Elements Control Nuclear Positioning and Asymmetric Cell Division during Physcomitrella patens Branching.

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.

Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens.

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.

Common-path multimodal three-dimensional fluorescence and phase imaging system.

A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach-Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss Physcomitrella patens. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

DIX Domain Polymerization Drives Assembly of Plant Cell Polarity Complexes.

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.

Kinesin-13 and Kinesin-8 Function during Cell Growth and Division in the Moss Physcomitrella patens.

Kinesin-13 and Kinesin-8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) Kinesin-13 have been shown to depolymerize MTs in vitro. However, the mitotic function of both kinesins has yet to be determined in plants. Here, we generated complete null mutants of Kinesin-13 and Kinesin-8 in moss (Physcomitrella patens). Both kinesins were found to be nonessential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either kinesin in vitro, while MT catastrophe-inducing activity (Kinesin-13) or MT gliding activity (Kinesin-8) was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and Kinesin-8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.

A versatile microfluidic device for highly inclined thin illumination microscopy in the moss Physcomitrella patens.

High-resolution microscopy is a valuable tool for studying cellular processes, such as signalling, membrane trafficking, or cytoskeleton remodelling. Several techniques of inclined illumination microscopy allow imaging at a near single molecular level; however, the application of these methods to plant cells is limited, owing to thick cell walls as well as the necessity to excise a part of the tissue for sample preparation. In this study, we utilised a simple, easy-to-use microfluidic device for highly inclined and laminated optical sheet (HILO) microscopy using a model plant Physcomitrella patens. We demonstrated that the shallow microfluidic device can be used for long-term culture of living cells and enables high-resolution HILO imaging of microtubules without perturbing their dynamics. In addition, our microdevice allows the supply and robust washout of compounds during HILO microscopy imaging, for example, to perform a microtubule regrowth assay. Furthermore, we tested long-term (48 h) HILO imaging using a microdevice and visualised the developmental changes in the microtubule dynamics during tissue regeneration. These novel applications of the microfluidic device provide a valuable resource for studying molecular dynamics in living plant cells.

Morphological and physiological traits in relation to carbon balance in a diverse clade of dryland mosses.

Plant functional trait analyses have focused almost exclusively on vascular plants, but bryophytes comprise ancient and diverse plant lineages that have widespread global distributions and important ecological functions in terrestrial ecosystems. We examined a diverse clade of dryland mosses, Syntrichia, and studied carbon balance during a precipitation event (C-balance), a functional trait related to physiological functioning, desiccation tolerance, survival, and ecosystem carbon and nitrogen cycling. We examined variability in C-balance among 14 genotypes of Syntrichia and measured an additional 10 physiological and 13 morphological traits at the cell, leaf, shoot, and clump level. C-balance varied 20-fold among genotypes, and highest C-balances were associated with long, narrow leaves with awns, and small cells with thick cell walls, traits that may influence water uptake and retention during a precipitation event. Ordination analyses revealed that the axis most strongly correlated with C-balance included the maximum chlorophyll fluorescence, Fm , indicating the importance of photosystem II health for C exchange. C-balance represents a key functional trait in bryophytes, but its measurement is time intensive and not feasible to measure on large scales. We propose two models (using physiological and morphological traits) to predict C-balance, whereby identifying simpler to measure traits for trait databases.

Involvement of Lhcb6 and Lhcb5 in Photosynthesis Regulation in Physcomitrella patens Response to Abiotic Stress.

There are a number of highly conserved photosystem II light-harvesting antenna proteins in moss whose functions are unclear. Here, we investigated the involvement of chlorophyll-binding proteins, Lhcb6 and Lhcb5, in light-harvesting and photosynthesis regulation in Physcomitrella patens. Lhcb6 or Lhcb5 knock-out resulted in a disordered thylakoid arrangement, a decrease in the number of grana membranes, and an increase in the number of starch granule. The absence of Lhcb6 or Lhcb5 did not noticeably alter the electron transport rates. However, the non-photochemical quenching activity in the lhcb5 mutant was dramatically reduced when compared to wild-type or lhcb6 plants under abiotic stress. Lhcb5 plants were more sensitive to photo-inhibition, while lhcb6 plants showed little difference compared to the wild-type plants under high-light stress. Moreover, both mutants showed a growth malformation phenotype with reduced chlorophyll content in the gametophyte. These results suggested that Lhcb6 or Lhcb5 played a unique role in plant development, thylakoid organization, and photoprotection of PSII in Physcomitrella, especially when exposed to high light or osmotic environments.

Automated Image Acquisition and Morphological Analysis of Cell Growth Mutants in Physcomitrella patens.

This protocol describes the automated imaging and a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. This method can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Furthermore, we describe how to acquire higher resolution images via the acquisition of a collection of multiple overlapping tiles from the same image. Information is presented to guide the investigator in the choice of vectors and basic conditions to perform transient RNA interference in moss. Detailed directions and examples for fluorescence image acquisition of small regenerating moss plants are provided. Instructions for stitching image tiles and for using an ImageJ-based macro for the quantitative morphological analysis of moss plants are also provided.

Stable Co-Cultivation of the Moss Physcomitrella patens with Human Cells in vitro as a New Approach to Support Metabolism of Diseased Alzheimer Cells.

Alzheimer's disease (AD) is a devastating slowly progressive neurodegenerative disorder with no cure. While there are many hypotheses, the exact mechanism causing this pathology is still unknown. Among many other features, AD is characterized by brain hypometabolism and decreased sugar availability, to which neurons eventually succumb. In light of this aspect of the disease, we hypothesized that boosting fuel supply to neurons may help them survive or at least alleviate some of the symptoms. Here we demonstrate that live moss Physcomitrella patens cells can be safely co-cultured with human fibroblasts in vitro and thus have a potential for providing human cells with energy and other vital biomolecules. These data may form the foundation for the development of novel approaches to metabolic bioengineering and treatment of diseased cells based on live plants. In addition, by providing alternative energy sources to human tissues, the biotechnological potential of this interkingdom setup could also serve as a springboard to foster innovative dietary processes addressing current challenges of mankind such as famine or supporting long-haul space flight.

The 2D to 3D growth transition in the moss Physcomitrella patens.

The colonization of land by plants coincided with and was most likely facilitated by the evolution of 3-dimensional (3D) growth. 3D growth is a pivotal feature of all land plants, but most develop in a way that precludes genetic investigation. In the moss Physcomitrella patens, 3D growth (gametophores) is preceded by an extended 2-dimensional (2D) growth phase (protonemata) that can be propagated indefinitely. Studies using P. patens have thus elucidated some of the molecular mechanisms underlying 3D growth regulation. This review summarizes the known molecular mechanisms underlying both the formation of gametophore initial cells and the development of the 3D growth in gametophores.

Cytological analysis and structural quantification of FtsZ1-2 and FtsZ2-1 network characteristics in Physcomitrella patens.

Although the concept of the cytoskeleton as a cell-shape-determining scaffold is well established, it remains enigmatic how eukaryotic organelles adopt and maintain a specific morphology. The Filamentous Temperature Sensitive Z (FtsZ) protein family, an ancient tubulin, generates complex polymer networks, with striking similarity to the cytoskeleton, in the chloroplasts of the moss Physcomitrella patens. Certain members of this protein family are essential for structural integrity and shaping of chloroplasts, while others are not, illustrating the functional diversity within the FtsZ protein family. Here, we apply a combination of confocal laser scanning microscopy and a self-developed semi-automatic computational image analysis method for the quantitative characterisation and comparison of network morphologies and connectivity features for two selected, functionally dissimilar FtsZ isoforms, FtsZ1-2 and FtsZ2-1. We show that FtsZ1-2 and FtsZ2-1 networks are significantly different for 8 out of 25 structural descriptors. Therefore, our results demonstrate that different FtsZ isoforms are capable of generating polymer networks with distinctive morphological and connectivity features which might be linked to the functional differences between the two isoforms. To our knowledge, this is the first study to employ computational algorithms in the quantitative comparison of different classes of protein networks in living cells.