Golgi clustering by the deficiency of COPI-SNARE in Drosophila photoreceptors.

A comprehensive study of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the fly genome by RNAi in Drosophila photoreceptors indicated that knockdown of any of the COPI-SNAREs, Syx18, Sec20, and Use1, resulted in the same characteristic phenotypes: Golgi stacks gathering on their trans-side, laterally expanded Golgi cisternae, and a reduced number of discrete Golgi stacks. These Golgi stacks are reminiscent of mammalian Golgi ribbons and Brefeldin A (BFA)-bodies in Drosophila S2 cells. As previously reported, BFA suppresses trans-Golgi network (TGN) fission and Golgi stack separation to form a BFA-body, which is a cluster of Golgi stacks cored by recycling endosomes. We found that the impairing each of COPI-SNAREs results in clustered Golgi stacks similar to BFA-bodies, indicating that COPI-SNAREs have a role to separate clustered Golgi stacks. These results further support the idea that the movement of Golgi stacks and the balance of fusion and fission of the TGN determine the level of clustering and ribbon formation of Golgi stacks within cells.

Chloroplasts with clefts and holes: a reassessment of the chloroplast shape using 3D FE-SEM cellular reconstruction of two species of Chlamydomonas.

Chloroplasts are usually considered spheroid organelles, but this is not the only shape of chloroplasts. The chloroplast of Chlamydomonas has been typically described as cup-shaped. However, in old studies, it was also modeled as a complex shape with "perforations" or windows. Here, we reconstructed the cellular architecture of Chlamydomonas reinhardtii and C. applanata using an array tomography system installed on a field emission scanning electron microscope. C. reinhardtii chloroplasts resembled a baseball glove or a cup without a side, featuring numerous large and small holes that may facilitate the transport of metabolites and proteins produced in the Golgi apparatus fitted in the holes. In a lipid-accumulating, high-light condition, the chloroplast volume increased by filling the side cleft with an entire wall. Many accumulated large lipid droplets were accommodated within the chloroplast holes, which could have been considered as "chloroplast lipid droplets." Mitochondrial meshworks surrounded the chloroplast. C. applanata chloroplasts appeared like a folded starfish or a cup with many side clefts and a few holes. There was a single mitochondrion or two that branched in a complex form. Tight contacts of various organelles were also found in C. applanata. These reconstructions illustrate the complexity of chloroplast shape, which necessitates a revised understanding of the localization of lipid droplets and the evolution of chloroplasts: The prevailing image of the spheroid chloroplasts that reminds us of the similarity between chloroplasts and cyanobacteria is no longer tenable.

E22G Aß40 fibril structure and kinetics illuminate how Aß40 rather than Aß42 triggers familial Alzheimer's.

Arctic (E22G) mutation in amyloid-ß (Aß enhances Aß40 fibril accumulation in Alzheimer's disease (AD). Unlike sporadic AD, familial AD (FAD) patients with the mutation exhibit more Aß40 in the plaque core. However, structural details of E22G Aß40 fibrils remain elusive, hindering therapeutic progress. Here, we determine a distinctive W-shaped parallel ß-sheet structure through co-analysis by cryo-electron microscopy (cryoEM) and solid-state nuclear magnetic resonance (SSNMR) of in-vitro-prepared E22G Aß40 fibrils. The E22G Aß40 fibrils displays typical amyloid features in cotton-wool plaques in the FAD, such as low thioflavin-T fluorescence and a less compact unbundled morphology. Furthermore, kinetic and MD studies reveal previously unidentified in-vitro evidence that E22G Aß40, rather than Aß42, may trigger Aß misfolding in the FAD, and prompt subsequent misfolding of wild-type (WT) Aß40/Aß42 via cross-seeding. The results provide insight into how the Arctic mutation promotes AD via Aß40 accumulation and cross-propagation.

Sandwich freezing and freeze-substitution of Arabidopsis plant tissues for electron microscopy.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two copper disks and has been used for observing exquisite close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed cultured cells, as well as animal and human tissues. In the present study, this method was applied to observe the fine structure of living Arabidopsis plant tissues and was found to achieve excellent ultrastructural preservation of cells and tissues. This is the first report of applying the sandwich freezing method to observe plant tissues.

Precocious cell differentiation occurs in proliferating cells in leaf primordia in Arabidopsis angustifolia3 mutant.

During leaf development, the timing of transition from cell proliferation to expansion is an important factor in determining the final organ size. However, the regulatory system involved in this transition remains less understood. To get an insight into this system, we investigated the compensation phenomenon, in which the cell number decreases while the cell size increases in organs with determinate growth. Compensation is observed in several plant species suggesting coordination between cell proliferation and expansion. In this study, we examined an Arabidopsis mutant of ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING FACTOR 1, a positive regulator of cell proliferation, which exhibits the compensation. Though the AN3 role has been extensively investigated, the mechanism underlying excess cell expansion in the an3 mutant remains unknown. Focusing on the early stage of leaf development, we performed kinematic, cytological, biochemical, and transcriptome analyses, and found that the cell size had already increased during the proliferation phase, with active cell proliferation in the an3 mutant. Moreover, at this stage, chloroplasts, vacuoles, and xylem cells developed earlier than in the wild-type cells. Transcriptome data showed that photosynthetic activity and secondary cell wall biosynthesis were activated in an3 proliferating cells. These results indicated that precocious cell differentiation occurs in an3 cells. Therefore, we suggest a novel AN3 role in the suppression of cell expansion/differentiation during the cell proliferation phase.

High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Effective alignment method using a diamond notch knife for correlative array tomography.

Correlative array tomography, combining light and electron microscopy via serial sections, plays a crucial role in the three-dimensional ultrastructural visualization and molecular distribution analysis in biological structures. To address the challenges of aligning fluorescence and electron microscopy images and aligning serial sections of irregularly shaped biological specimens, we developed a diamond notch knife, a new tool for puncturing holes using a diamond needle. The diamond needle featured a triangular and right-angled tip, enabling the drilling of deep holes upon insertion into the polished block face. This study describes the application of the diamond notch knife in correlative array tomography.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Petal abscission is promoted by jasmonic acid-induced autophagy at Arabidopsis petal bases.

In angiosperms, the transition from floral-organ maintenance to abscission determines reproductive success and seed dispersion. For petal abscission, cell-fate decisions specifically at the petal-cell base are more important than organ-level senescence or cell death in petals. However, how this transition is regulated remains unclear. Here, we identify a jasmonic acid (JA)-regulated chromatin-state switch at the base of Arabidopsis petals that directs local cell-fate determination via autophagy. During petal maintenance, co-repressors of JA signaling accumulate at the base of petals to block MYC activity, leading to lower levels of ROS. JA acts as an airborne signaling molecule transmitted from stamens to petals, accumulating primarily in petal bases to trigger chromatin remodeling. This allows MYC transcription factors to promote chromatin accessibility for downstream targets, including NAC DOMAIN-CONTAINING PROTEIN102 (ANAC102). ANAC102 accumulates specifically at the petal base prior to abscission and triggers ROS accumulation and cell death via AUTOPHAGY-RELATED GENEs induction. Developmentally induced autophagy at the petal base causes maturation, vacuolar delivery, and breakdown of autophagosomes for terminal cell differentiation. Dynamic changes in vesicles and cytoplasmic components in the vacuole occur in many plants, suggesting JA-NAC-mediated local cell-fate determination by autophagy may be conserved in angiosperms.

Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease.

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control.

The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha.

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.

Microtubule-associated phase separation of MIDD1 tunes cell wall spacing in xylem vessels in Arabidopsis thaliana.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

A cell wall-localized cytokinin/purine riboside nucleosidase is involved in apoplastic cytokinin metabolism in Oryza sativa.

In the final step of cytokinin biosynthesis, the main pathway is the elimination of a ribose-phosphate moiety from the cytokinin nucleotide precursor by phosphoribohydrolase, an enzyme encoded by a gene named LONELY GUY (LOG). This reaction accounts for most of the cytokinin supply needed for regulating plant growth and development. In contrast, the LOG-independent pathway, in which dephosphorylation and deribosylation sequentially occur, is also thought to play a role in cytokinin biosynthesis, but the gene entity and physiological contribution have been elusive. In this study, we profiled the phytohormone content of chromosome segment substitution lines of Oryza sativa and searched for genes affecting the endogenous levels of cytokinin ribosides by quantitative trait loci analysis. Our approach identified a gene encoding an enzyme that catalyzes the deribosylation of cytokinin nucleoside precursors and other purine nucleosides. The cytokinin/purine riboside nucleosidase 1 (CPN1) we identified is a cell wall-localized protein. Loss-of-function mutations (cpn1) were created by inserting a Tos17-retrotransposon that altered the cytokinin composition in seedling shoots and leaf apoplastic fluid. The cpn1 mutation also abolished cytokinin riboside nucleosidase activity in leaf extracts and attenuated the trans-zeatin riboside-responsive expression of cytokinin marker genes. Grain yield of the mutants declined due to altered panicle morphology under field-grown conditions. These results suggest that the cell wall-localized LOG-independent cytokinin activating pathway catalyzed by CPN1 plays a role in cytokinin control of rice growth. Our finding broadens our spatial perspective of the cytokinin metabolic system.

Seasonal gene expression signatures of delayed fertilization in Fagaceae.

In the family Fagaceae, fertilization is delayed by several weeks to 1 year after pollination, leading to 1- or 2-year fruiting species depending on whether fruiting occurs in the same or the next year after flowering. To investigate physiological responses underlying the regulation of delayed fertilization, we monitored seasonal changes in genome-wide gene expression in tissues including leaves and buds over 2 years under natural conditions in one- (Quercus glauca) and 2-year fruiting species (Lithocarpus edulis). Genes associated with metabolic changes in response to winter cold, photosynthesis and cell proliferation, which are essential for survival and growth, showed highly conserved seasonal expression profiles between species. However, seasonal expression profiles diverged between species in genes associated with pollination, an important process contributing to the origin and maintenance of the reproductive barrier between plant species. By comparing seasonal progression of ovule development and gene expression in pistillate flowers, we revealed that ovules started developing after winter in the 2-year fruiting species, which could be linked to the activation of genes involved in fertilization and female gametophyte development after winter. These findings suggest that the 2-year fruiting species may have evolved a requirement of winter cold to prevent fertilization before winter and facilitate fertilization and embryo development in the following spring when temperature rises. This study offers new possibilities to explore the evolution of reproductive strategies in Fagaceae.

Starch-dependent sodium accumulation in the leaves of Vigna riukiuensis.

This research provides insight into a unique salt tolerance mechanism of Vigna riukiuensis. V. riukiuensis is one of the salt-tolerant species identified from the genus Vigna. We have previously reported that V. riukiuensis accumulates a higher amount of sodium in the leaves, whereas V. nakashimae, a close relative of V. riukiuensis, suppresses sodium allocation to the leaves. We first suspected that V. riukiuensis would have developed vacuoles for sodium sequestration, but there were no differences compared to a salt-sensitive species V. angularis. However, many starch granules were observed in the chloroplasts of V. riukiuensis. In addition, forced degradation of leaf starch by shading treatment resulted in no radio-Na (22Na) accumulation in the leaves. We performed SEM-EDX to locate Na in leaf sections and detected Na in chloroplasts of V. riukiuensis, especially around the starch granules but not in the middle of. Our results could provide the second evidence of the Na-trapping system by starch granules, following the case of common reed that accumulates starch granule at the shoot base for binding Na.

TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the ß-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport ß-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel.

The conserved nine-fold structural symmetry of the centriole is thought to be generated by cooperation between two mechanisms, one dependent on and the other independent of the cartwheel, a sub-centriolar structure consisting of a hub and nine spokes. However, the molecular entity of the cartwheel-independent mechanism has not been elucidated. Here, using Chlamydomonas reinhardtii mutants, we show that Bld10p/Cep135, a conserved centriolar protein that connects cartwheel spokes and triplet microtubules, plays a central role in this mechanism. Using immunoelectron microscopy, we localized hemagglutinin epitopes attached to distinct regions of Bld10p along two lines that connect adjacent triplets. Consistently, conventional and cryo-electron microscopy identified crosslinking structures at the same positions. In centrioles formed in the absence of the cartwheel, truncated Bld10p was found to significantly reduce the inter-triplet distance and frequently form eight-microtubule centrioles. These results suggest that the newly identified crosslinks are comprised of part of Bld10p/Cep135. We propose that Bld10p determines the inter-triplet distance in the centriole and thereby regulates the number of triplets in a cartwheel-independent manner.