The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens.

A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.

Surface-localized glycoproteins act through class C ARFs to fine-tune gametophore initiation in Physcomitrium patens.

Arabinogalactan proteins are functionally diverse cell wall structural glycoproteins that have been implicated in cell wall remodeling, although the mechanistic actions remain elusive. Here, we identify and characterize two AGP glycoproteins, SLEEPING BEAUTY (SB) and SB-like (SBL), that negatively regulate the gametophore bud initiation in Physcomitrium patens by dampening cell wall loosening/softening. Disruption of SB and SBL led to accelerated gametophore formation and altered cell wall compositions. The function of SB is glycosylation dependent and genetically connected with the class C auxin response factor (ARF) transcription factors PpARFC1B and PpARFC2. Transcriptomics profiling showed that SB upregulates PpARFC2, which in turn suppresses a range of cell wall-modifying genes that are required for cell wall loosening/softening. We further show that PpARFC2 binds directly to multiple AuxRE motifs on the cis-regulatory sequences of PECTIN METHYLESTERASE to suppress its expression. Hence, our results demonstrate a mechanism by which the SB modulates the strength of intracellular auxin signaling output, which is necessary to fine-tune the timing of gametophore initials formation.

The bryophytes Physcomitrium patens and Marchantia polymorpha as model systems for studying evolutionary cell and developmental biology in plants.

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.

Characterisation of rapid alkalinisation factors in Physcomitrium patens reveals functional conservation in tip growth.

Small signalling peptides are key molecules for cell-to-cell communications in plants. The cysteine-rich signalling peptide, rapid alkalinisation factors (RALFs) family are involved in diverse developmental and stress responses and have expanded considerably during land plant evolution, implying neofunctionalisations in the RALF family. However, the ancestral roles of RALFs when land plant first acquired them remain unknown. Here, we functionally characterised two of the three RALFs in bryophyte Physcomitrium patens using loss-of-function mutants, overexpressors, as well as fluorescent proteins tagged reporter lines. We showed that PpRALF1 and PpRALF2 have overlapping functions in promoting protonema tip growth and elongation, showing a homologous function as the Arabidopsis RALF1 in promoting root hair tip growth. Although both PpRALFs are secreted to the plasma membrane on which PpRALF1 symmetrically localised, PpRALF2 showed a polarised localisation at the growing tip. Notably, proteolytic cleavage of PpRALF1 is necessary for its function. Our data reveal a possible evolutionary origin of the RALF functions and suggest that functional divergence of RALFs is essential to drive complex morphogenesis and to facilitate other novel processes in land plants.

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.

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

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.

AP2/ERF transcription factors regulate salt-induced chloroplast division in the moss Physcomitrella patens.

Chloroplast division is a critical process for the maintenance of appropriate chloroplast number in plant cells. It is known that in some plant species and cell types, environmental stresses can affect chloroplast division, differentiation and morphology, however the significance and regulation of these processes are largely unknown. Here we investigated the regulation of salt stress-induced chloroplast division in protonemal cells of the moss, Physcomitrella patens, and found that, salt stress as one of the major abiotic stresses, induced chloroplast division and resulted in increased chloroplast numbers. We further identified three APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors (TFs) that were responsible for this regulation. These AP2/ERF genes were up-regulated under salt stress, and amino acid sequences and phylogenetic analyses indicated that all TFs possess only one conserved AP2 domain and likely belong to the same subgroup of ERF-B3 in the AP2/ERF superfamily. Overexpression of these TFs significantly increased the chloroplast number even in the absence of NaCl stress. On the contrary, inducible overexpression of the dominant repressor form of these TFs suppressed salt stress-induced chloroplast division. Thus, our results suggest that salt stress induced-chloroplast division is regulated through members of the AP2/ERF TF superfamily.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.

The PSI-PSII Megacomplex in Green Plants.

Energy dissipation is crucial for land and shallow-water plants exposed to direct sunlight. Almost all green plants dissipate excess excitation energy to protect the photosystem reaction centers, photosystem II (PSII) and photosystem I (PSI), and continue to grow under strong light. In our previous work, we reported that about half of the photosystem reaction centers form a PSI-PSII megacomplex in Arabidopsis thaliana, and that the excess energy was transferred from PSII to PSI fast. However, the physiological function and structure of the megacomplex remained unclear. Here, we suggest that high-light adaptable sun-plants accumulate the PSI-PSII megacomplex more than shade-plants. In addition, PSI of sun-plants has a deep trap to receive excitation energy, which is low-energy chlorophylls showing fluorescence maxima longer than 730 nm. This deep trap may increase the high-light tolerance of PSI by improving excitation energy dissipation. Electron micrographs suggest that one PSII dimer is directly sandwiched between two PSIs with 2-fold rotational symmetry in the basic form of the PSI-PSII megacomplex in green plants. This structure should enable fast energy transfer from PSII to PSI and allow energy in PSII to be dissipated via the deep trap in PSI.

Abscisic Acid Acts as a Regulator of Molecular Trafficking through Plasmodesmata in the Moss Physcomitrella patens.

In multi-cellular organisms, cell-to-cell communication is crucial for adapting to changes in the surrounding environment. In plants, plasmodesmata (PD) provide a unique pathway for cell-to-cell communication. PD interconnect most cells and generate a cytoplasmic continuum, allowing the trafficking of various micro- and macromolecules between cells. This molecular trafficking through PD is dynamically regulated by altering PD permeability dependent on environmental changes, thereby leading to an appropriate response to various stresses; however, how PD permeability is dynamically regulated is still largely unknown. Moreover, studies on the regulation of PD permeability have been conducted primarily in a limited number of angiosperms. Here, we studied the regulation of PD permeability in the moss Physcomitrella patens and report that molecular trafficking through PD is rapidly and reversibly restricted by abscisic acid (ABA). Since ABA plays a key role in various stress responses in the moss, PD permeability can be controlled by ABA to adapt to surrounding environmental changes. This ABA-dependent restriction of PD trafficking correlates with a reduction in PD pore size. Furthermore, we also found that the rate of macromolecular trafficking is higher in an ABA-synthesis defective mutant, suggesting that the endogenous level of ABA is also important for PD-mediated macromolecular trafficking. Thus, our study provides compelling evidence that P. patens exploits ABA as one of the key regulators of PD function.

Formation of a PSI-PSII megacomplex containing LHCSR and PsbS in the moss Physcomitrella patens.

Mosses are one of the earliest land plants that diverged from fresh-water green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.

Correction to: A hypergravity environment increases chloroplast size, photosynthesis, and plant growth in the moss Physcomitrella patens.

The original article can be found online.

PCoM-DB Update: A Protein Co-Migration Database for Photosynthetic Organisms.

The identification of protein complexes is important for the understanding of protein structure and function and the regulation of cellular processes. We used blue-native PAGE and tandem mass spectrometry to identify protein complexes systematically, and built a web database, the protein co-migration database (PCoM-DB, http://pcomdb.lowtem.hokudai.ac.jp/proteins/top), to provide prediction tools for protein complexes. PCoM-DB provides migration profiles for any given protein of interest, and allows users to compare them with migration profiles of other proteins, showing the oligomeric states of proteins and thus identifying potential interaction partners. The initial version of PCoM-DB (launched in January 2013) included protein complex data for Synechocystis whole cells and Arabidopsis thaliana thylakoid membranes. Here we report PCoM-DB version 2.0, which includes new data sets and analytical tools. Additional data are included from whole cells of the pelagic marine picocyanobacterium Prochlorococcus marinus, the thermophilic cyanobacterium Thermosynechococcus elongatus, the unicellular green alga Chlamydomonas reinhardtii and the bryophyte Physcomitrella patens. The Arabidopsis protein data now include data for intact mitochondria, intact chloroplasts, chloroplast stroma and chloroplast envelopes. The new tools comprise a multiple-protein search form and a heat map viewer for protein migration profiles. Users can compare migration profiles of a protein of interest among different organelles or compare migration profiles among different proteins within the same sample. For Arabidopsis proteins, users can compare migration profiles of a protein of interest with putative homologous proteins from non-Arabidopsis organisms. The updated PCoM-DB will help researchers find novel protein complexes and estimate their evolutionary changes in the green lineage.

A hypergravity environment increases chloroplast size, photosynthesis, and plant growth in the moss Physcomitrella patens.

The physiological and anatomical responses of bryophytes to altered gravity conditions will provide crucial information for estimating how plant physiological traits have evolved to adapt to significant increases in the effects of gravity in land plant history. We quantified changes in plant growth and photosynthesis in the model plant of mosses, Physcomitrella patens, grown under a hypergravity environment for 25 days or 8 weeks using a custom-built centrifuge equipped with a lighting system. This is the first study to examine the response of bryophytes to hypergravity conditions. Canopy-based plant growth was significantly increased at 10×g, and was strongly affected by increases in plant numbers. Rhizoid lengths for individual gametophores were significantly increased at 10×g. Chloroplast diameters (major axis) and thicknesses (minor axis) in the leaves of P. patens were also increased at 10×g. The area-based photosynthesis rate of P. patens was also enhanced at 10×g. Increases in shoot numbers and chloroplast sizes may elevate the area-based photosynthesis rate under hypergravity conditions. We observed a decrease in leaf cell wall thickness under hypergravity conditions, which is in contrast to previous findings obtained using angiosperms. Since mosses including P. patens live in dense populations, an increase in canopy-based plant numbers may be effective to enhance the toughness of the population, and, thus, represents an effective adaptation strategy to a hypergravity environment for P. patens.

A model system for analyzing intercellular communication through plasmodesmata using moss protonemata and leaves.

Plant growth, development, and environmental responses require the proper regulation of intercellular movement of signals and nutrients. For this, plants have specialized cytoplasmic channels, the plasmodesmata (PD), which allow the symplasmic movement of micro- and macromolecules between neighboring cells. Internal and external signals spatio-temporally regulate the movement of molecules through the PD to control plant development and environmental responses. Although some aspects of targeted movement of molecules have been revealed, the mechanisms of non-targeted, diffusible flow of molecules through PD, and its regulation and function, remain poorly understood, particularly at the cellular level. Previously, we developed a system to quantitatively analyze non-targeted movement of a photoconvertible fluorescent protein, Dendra2, at the single-cell level in the filamentous protonemata tissue of the moss Physcomitrella patens. In protonemata, one-dimensional intercellular communication can be easily observed and quantitatively analyzed at the cellular level. In this review, we describe how protonemata and leaves of P. patens can be used to study symplasmic movement through PD, and discuss how this system can help improve our understanding of PD regulation and function in development and environmental responses in plants.

Unraveling the Mechanism of Cork Spot-like Physiological Disorders in 'Kurenainoyume' Apples Based on Occurrence Location.

Cork spot-like physiological disorder (CSPD) is a newly identified issue in 'Kurenainoyume' apples, yet its mechanism remains unclear. To investigate CSPD, we conducted morphological observations on 'Kurenainoyume' apples with and without pre-harvest fruit-bagging treatment using light-impermeable paper bags. Non-bagged fruit developed CSPD in mid-August, while no CSPD symptoms were observed in bagged fruit. The bagging treatment significantly reduced the proportion of opened lenticels, with only 17.9% in bagged fruit compared to 52.0% in non-bagged fruits. In non-bagged fruit, CSPD spots tended to increase from the lenticels, growing in size during fruit development. The cuticular thickness and cross-sectional area of fresh cells in CSPD spots were approximately 16 µm and 1600 µm², respectively. Healthy non-bagged fruit reached these values around 100 to 115 days after full bloom from mid- to late August. Microscopic and computerized tomography scanning observations revealed that many CSPD spots developed at the tips of vascular bundles. Therefore, CSPD initiation between opened lenticels and vascular bundle tips may be influenced by water stress, which is potentially caused by water loss, leading to cell death and the formation of CSPD spots.

Quantitative imaging of directional transport through plasmodesmata in moss protonemata via single-cell photoconversion of Dendra2.

Cell-to-cell transport of molecules in plants must be properly regulated for plant growth and development. One specialized mechanism that plants have evolved involves transport through plasmodesmata (PD), but when and how transport of molecules via PD is regulated among individual cells remains largely unknown, particularly at the single-cell level. Here, we developed a tool for quantitatively analyzing cell-to-cell transport via PD at a single-cell level using protonemata of Physcomitrella patens and a photoconvertible fluorescent protein, Dendra2. In the filamentous protonemal tissues, one-dimensional intercellular communication can be observed easily. Using this system, we found that Dendra2 was directionally transported toward the apex of the growing protonemata. However, this directional transport could be eliminated by incubation in the dark or treatment with a metabolic inhibitor. Thus, we propose that directional transport of macromolecules can occur via PD in moss protonemata, and may be affected by the photosynthetic and metabolic activity of cells.

ABA signaling converts stem cell fate by substantiating a tradeoff between cell polarity, growth and cell cycle progression and abiotic stress responses in the moss Physcomitrium patens.

Abscisic acid (ABA)-mediated abiotic stress tolerance causes plant growth inhibition. Under such stress conditions, some mosses generate de novo stress-resistant stem cells, also called brood cells or brachycytes, that do not exist under normal conditions. However, the cell physiological basis of the growth inhibition and the stem cell formation is not well understood. Here, we show that the ABA-induced growth inhibition of the moss Physcomitrium patens apical protonemal cells (protonemal stem cells) is mediated through a shift from asymmetric to symmetric cell division. This change of the cell division mode, and consequently change of stem cell activity, is substantiated by dampening cell polarity and cell proliferative activity through the altered distribution of cytoskeletal elements, the mitotic spindle and the vacuole, which results in the production of stress-resistant stem cells. Alteration of the cell physiological data is supported by the results of RNAseq analysis indicating rapid changes in both cell polarity and cell cycle regulation, while long-term treatments with ABA for 5 to 10 days impact mainly the transcriptional and translational regulation. The regulation of cell polarity and cell cycle genes suggests growth arrest mediated by small GTPases (ROPs) and their guanine exchange factors (ROPGEFs) and by cyclin and cyclin-dependent-kinase complex, respectively. Our data suggest that a tradeoff relationship between growth ability and abiotic stress response in the moss is substantiated by ABA signaling to suppress cell polarity and asymmetric cell growth and may play a pivotal role in stem cell fate conversion to newly produced stress-resistant stem cells.