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.

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.

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.

Three-dimensionally visualized rhizoid system of moss, Physcomitrium patens, by refraction-contrast X-ray micro-computed tomography.

Land plants have two types of shoot-supporting systems, root system and rhizoid system, in vascular plants and bryophytes. However, since the evolutionary origin of the systems is different, how much they exploit common systems or distinct systems to architect their structures is largely unknown. To understand the regulatory mechanism of how bryophytes architect the rhizoid system responding to environmental factors, we have developed the methodology to visualize and quantitatively analyze the rhizoid system of the moss, Physcomitrium patens, in 3D. The rhizoids having a diameter of 21.3 µm on the average were visualized by refraction-contrast X-ray micro-computed tomography using coherent X-ray optics available at synchrotron radiation facility SPring-8. Three types of shape (ring-shape, line and black circle) observed in tomographic slices of specimens embedded in paraffin were confirmed to be the rhizoids by optical and electron microscopy. Comprehensive automatic segmentation of the rhizoids, which appeared in three different form types in tomograms, was tested by a method using a Canny edge detector or machine learning. The accuracy of output images was evaluated by comparing with the manually segmented ground truth images using measures such as F1 score and Intersection over Union, revealing that the automatic segmentation using machine learning was more effective than that using the Canny edge detector. Thus, machine learning-based skeletonized 3D model revealed quite dense distribution of rhizoids. We successfully visualized the moss rhizoid system in 3D for the first time.

Abscisic acid switches cell division modes of asymmetric cell division and symmetric cell division in stem cells of protonemal filaments in the moss Physcomitrium patens.

Multicellular organisms regulate cell numbers and cell fate by using asymmetric cell division (ACD) and symmetric cell division (SCD) during their development and to adapt to unfavorable environmental conditions. A stem cell self-renews and generates differentiated cells. In plants, various types of cells are produced by ACD or SCD; however, the molecular mechanisms of ACD or SCD and the cell division mode switch are largely unknown. The moss Physcomitrium (Physcomitrella) patens is a suitable model to study plant stem cells due to its simple anatomy. Here, we report the cell division mode switch induced by abscisic acid (ABA) in P. patens. ABA is synthesized in response to abiotic stresses and induces round-shape cells, called brood cells, from cylindrical protonemal cells. Although two daughter cells with distinct sizes were produced by ACD in a protonemal stem cell on ABA-free media, the sizes of two daughter cells became similar with ABA treatment. Actin microfilaments were spatially localized on the apices of apical stem cells in protonemata on ABA-free media, but the polar accumulation was lost under the condition of ABA treatment. Moreover, ABA treatment conferred an identical cell fate to the daughter cells in terms of cell division activity. Collectively, the results indicate ABA may suppress the ACD characteristics but evoke SCD in cells. We also noticed that ABA-induced brood cells not only self-renewed but regenerated protonemal cells when ABA was removed from the media, suggesting that brood cells are novel stem cells that are induced by environmental signals in P. patens.

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.

Callose Detection and Quantification at Plasmodesmata in Bryophytes.

In bryophytes (i.e., mosses, liverworts, and hornworts), extant representatives of early land plants, plasmodesmata have been described in a wide range of tissues. Although their contribution to bryophyte morphogenesis remains largely unexplored, several recent studies have suggested that the deposition of callose around plasmodesmata might regulate developmental and physiological responses in mosses. In this chapter, we provide a protocol to image and quantify callose levels in the filamentous body of the model moss Physcomitrium (Physcomitrella) patens and discuss possible alternatives and pitfalls. More generally, this protocol establishes a framework to explore the distribution of callose in other bryophytes.

Tracking Intercellular Movement of Fluorescent Proteins in Bryophytes.

An important approach to investigate intercellular connectivity via plasmodesmata is to visualize and track the movement of fluorescent proteins between cells. The intercellular connectivity is largely controlled by the size exclusion limit of the pores. Over the past few decades, the technique to observe and analyze intercellular movement of a fluorescent protein has been developed mainly in angiosperms such as Arabidopsis thaliana. We recently applied the corresponding system to track the intercellular movement of the fluorescent protein Dendra2 in the moss Physcomitrium (Physcomitrella) patens. The protonemal tissues are particularly suited for observation of the intercellular movement due to the simple organization. Here, we describe a protocol suitable for the analysis of Dendra2 movement between cells in P. patens.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

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.

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.

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.

Metabolic Control of Gametophore Shoot Formation through Arginine in the Moss Physcomitrium patens.

Shoot formation is accompanied by active cell proliferation and expansion, requiring that metabolic state adapts to developmental control. Despite the importance of such metabolic reprogramming, it remains unclear how development and metabolism are integrated. Here, we show that disruption of ANGUSTIFOLIA3 orthologs (PpAN3s) compromises gametophore shoot formation in the moss Physcomitrium patens due to defective cell proliferation and expansion. Trans-omics analysis reveals that the downstream activity of PpAN3 is linked to arginine metabolism. Elevating arginine level by chemical treatment leads to stunted gametophores and causes Ppan3 mutant-like transcriptional changes in the wild-type plant. Furthermore, ectopic expression of AtAN3 from Arabidopsis thaliana ameliorates the defective arginine metabolism and promotes gametophore formation in Ppan3 mutants. Together, these findings indicate that arginine metabolism is a key pathway associated with gametophore formation and provide evolutionary insights into the establishment of the shoot system in land plants through the integration of developmental and metabolic processes.

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.

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.

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.

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.