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Nicotine is a major alkaloid accumulatmg in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATEI and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells. [ABSTRACT FROM AUTHOR]

We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are β-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research. [Copyright &y& Elsevier]

Syringic acid, a phenolic compound, serves a variety of beneficial functions in cells. Syringic acid increases in plants in response to cesium, and exogenous application of syringic acid resulted in a significant attenuation of cesium-induced growth defects in Arabidopsis. In addition, cesium or syringic acid application to plants also resulted in increased lignin deposition in interfascicular fibers. To better understand the role of lignin and syringic acid in attenuating cesium-induced growth defects, two mutants for ArabidopsisREDUCED EPIDERMAL FLUORESCENE 4 (REF4) and fourteen laccase mutants, some of which have lower levels of lignin, were evaluated for their response to cesium. These mutants responded differently to cesium stress, compared to control plants, and the application of syringic acid alleviated cesium-induced growth defects in the laccase mutants but not in the ref4 mutants. These findings imply that lignin plays a role in cesium signaling but the attenuation of cesium stress defects by syringic acid is mediated by regulatory components of lignin biosynthesis and not lignin biosynthesis itself. In contrast, syringic acid did not alleviate any low potassium-induced growth defects. Collectively, our findings provide the first established link between lignin and cesium stress via syringic acid in plants. [ABSTRACT FROM AUTHOR]

Arctic (E22G) mutation in amyloid-β (Aβ enhances Aβ40 fibril accumulation in Alzheimer's disease (AD). Unlike sporadic AD, familial AD (FAD) patients with the mutation exhibit more Aβ40 in the plaque core. However, structural details of E22G Aβ40 fibrils remain elusive, hindering therapeutic progress. Here, we determine a distinctive W-shaped parallel β-sheet structure through co-analysis by cryo-electron microscopy (cryoEM) and solid-state nuclear magnetic resonance (SSNMR) of in-vitro-prepared E22G Aβ40 fibrils. The E22G Aβ40 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 Aβ40, rather than Aβ42, may trigger Aβ misfolding in the FAD, and prompt subsequent misfolding of wild-type (WT) Aβ40/Aβ42 via cross-seeding. The results provide insight into how the Arctic mutation promotes AD via Aβ40 accumulation and cross-propagation., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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

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., Competing Interests: The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ezaki, Koga, Takeda-Kamiya, Toyooka, Higaki, Sakamoto and Tsukaya.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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 CO 2 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., (© 2024. The Author(s).)

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 Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

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., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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 ( 22 Na) 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., (© 2023. The Author(s).)

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 β-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 β-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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mobilisation of seed storage reserves is important for seedling establishment in Arabidopsis . In this process, sucrose is synthesised from triacylglycerol via core metabolic processes. Mutants with defects in triacylglycerol-to-sucrose conversion display short etiolated seedlings. We found that whereas sucrose content in the indole-3-butyric acid response 10 ( ibr10 ) mutant was significantly reduced, hypocotyl elongation in the dark was unaffected, questioning the role of IBR10 in this process. To dissect the metabolic complexity behind cell elongation, a quantitative-based phenotypic analysis combined with a multi-platform metabolomics approach was applied. We revealed that triacylglycerol and diacylglycerol breakdown were disrupted in ibr10 , resulting in low sugar content and poor photosynthetic ability. Importantly, batch-learning self-organised map clustering revealed that threonine level was correlated with hypocotyl length. Consistently, exogenous threonine supply stimulated hypocotyl elongation, indicating that sucrose levels are not always correlated with etiolated seedling length, suggesting the contribution of amino acids in this process., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2022.)

Different organelles function coordinately in numerous intracellular processes. Photorespiration incidental to photosynthetic carbon fixation is organized across three subcellular compartments: chloroplasts, peroxisomes, and mitochondria. Under light conditions, these three organelles often form a ternary organellar complex in close proximity, suggesting a connection with metabolism during photorespiration. However, due to the heterogeneity of intercellular organelle localization and morphology, organelles' responses to changes in the external environment remain poorly understood. Here, we used array tomography by field emission scanning electron microscopy to image organelles inside the whole plant cell at nanometer resolution, generating a three-dimensional (3D) spatial map of the light-dependent positioning of chloroplasts, peroxisomes, nuclei, and vacuoles. Our results show, in light-treated cells, the volume of peroxisomes increased, and mitochondria were simplified. In addition, the population of free organelles decreased, and the ternary complex centered on chloroplasts increased. Moreover, our results emphasized the expansion of the proximity area rather than the increase in the number of proximity sites interorganelles. All of these phenomena were quantified for the first time on the basis of nanoscale spatial maps. In summary, we provide the first 3D reconstruction of Arabidopsis mesophyll cells, together with nanoscale quantified organelle morphology and their positioning via proximity areas, and then evidence of their light-dependent changes., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Highly efficient tissue repair is pivotal for surviving damage-associated stress. Plants generate callus upon injury to heal wound sites, yet regulatory mechanisms of tissue repair remain elusive. Here, we identified WUSCHEL-RELATED HOMEOBOX 13 (WOX13) as a key regulator of callus formation and organ adhesion in Arabidopsis (Arabidopsis thaliana). WOX13 belongs to an ancient subclade of the WOX family, and a previous study shows that WOX13 orthologs in the moss Physcomitrium patens (PpWOX13L) are involved in cellular reprogramming at wound sites. We found that the Arabidopsis wox13 mutant is totally defective in establishing organ reconnection upon grafting, suggesting that WOX13 is crucial for tissue repair in seed plants. WOX13 expression rapidly induced upon wounding, which was partly dependent on the activity of an AP2/ERF transcription factor, WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1). WOX13 in turn directly upregulated WIND2 and WIND3 to further promote cellular reprogramming and organ regeneration. We also found that WOX13 orchestrates the transcriptional induction of cell wall-modifying enzyme genes, such as GLYCOSYL HYDROLASE 9Bs, PECTATE LYASE LIKEs and EXPANSINs. Furthermore, the chemical composition of cell wall monosaccharides was markedly different in the wox13 mutant. These data together suggest that WOX13 modifies cell wall properties, which may facilitate efficient callus formation and organ reconnection. Furthermore, we found that PpWOX13L complements the Arabidopsis wox13 mutant, suggesting that the molecular function of WOX13 is partly conserved between mosses and seed plants. This study provides key insights into the conservation and functional diversification of the WOX gene family during land plant evolution., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Duckweeds ( Araceae: Lemnoideae ) are aquatic monocotyledonous plants that are characterized by their small size, rapid growth, and wide distribution. Developmental processes regulating the formation of their small leaf-like structures, called fronds, and tiny flowers are not well characterized. In many plant species, flowering is promoted by the florigen activation complex, whose major components are florigen FLOWERING LOCUS T (FT) protein and transcription factor FD protein. How this complex is regulated at the molecular level during duckweed flowering is also not well understood. In this study, we characterized the course of developmental changes during frond development and flower formation in Lemna aequinoctialis Nd, a short-day plant. Detailed observations of frond and flower development revealed that cell proliferation in the early stages of frond development is active as can be seen in the separate regions corresponding to two budding pouches in the proximal region of the mother frond. L. aequinoctialis produces two stamens of different lengths with the longer stamen growing more rapidly. Using high-throughput RNA sequencing (RNA-seq) and de novo assembly of transcripts from plants induced to flower, we identified the L. aequinoctialis FT and FD genes, whose products in other angiosperms form a transcriptional complex to promote flowering. We characterized the protein-protein interaction of duckweed FT and FD in yeast and examined the functions of the two gene products by overexpression in Arabidopsis . We found that L. aequinoctialis FTL1 promotes flowering, whereas FTL2 suppresses flowering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yoshida, Taoka, Hosaka, Tanaka, Kobayashi, Muranaka, Toyooka, Oyama and Tsuji.)

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus ( Asparagus officinalis ). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis., (© 2021 Japanese Society for Plant Biotechnology.)

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus , we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used 15 N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the 15 N-labeled and non-labeled MS/MS spectra., (© 2021 Japanese Society for Plant Biotechnology.)

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO 2 environment predicted for the near future will reduce nitrate utilization by C 3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress., (© 2021. The Author(s).)

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca 2+ - and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca 2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca 2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis., Competing Interests: The authors declare no competing interest.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

The cyanobacterium Acaryochloris marina MBIC 11017 (A. marina 11017) possesses chlorophyll d (Chl. d) peaking at 698 nm as photosystem reaction center pigments, instead of chlorophyll a (Chl. a) peaking at 665 nm. About 95% of the total chlorophylls is Chl. d in A. marina 11017. In addition, A. marina 11017 possesses phycobilisome (PBS) supercomplex to harvest orange light and to transfer the absorbing energy to the photosystems. In this context, A. marina 11017 utilizes both far-red and orange light as the photosynthetic energy source. In the present study, we incubated A. marina 11017 cells under monochromatic orange and far-red light conditions and performed transcriptional and morphological studies by RNA-seq analysis and electron microscopy. Cellular absorption spectra, transcriptomic profiles, and microscopic observations demonstrated that PBS was highly accumulated under an orange light condition relative to a far-red light condition. Notably, transcription of one cpcBA operon encoding the phycobiliprotein of the phycocyanin was up-regulated under the orange light condition, but another operon was constitutively expressed under both conditions, indicating functional diversification of these two operons for light harvesting. Taking the other observations into consideration, we could illustrate the photoacclimation processes of A. marina 11017 in response to orange and far-red light conditions in detail.

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with 15 nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus . Principal component analysis using 15 N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with 15 N. Paring of the 15 N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m / z value, and a nonlabeled product ion at m / z 144.08 that indicates an indolic skeleton. The mass shift of the m / z value of the product and precursor ions to their 15 N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.

Patterned cell wall deposition is crucial for cell shapes and functions. In Arabidopsis xylem vessels, ROP11 GTPase locally inhibits cell wall deposition through microtubule disassembly, inducing pits in cell walls. Here, we show that an additional ROP signaling pathway promotes cell wall growth at pit boundaries. Two proteins, Boundary of ROP domain1 (BDR1) and Wallin (WAL), localize to pit boundaries and regulate cell wall growth. WAL interacts with F-actin and promotes actin assembly at pit boundaries while BDR1 is a ROP effector. BDR1 interacts with WAL, suggesting that WAL could be recruited to the plasma membrane by a ROP-dependent mechanism. These results demonstrate that BDR1 and WAL mediate a ROP-actin pathway that shapes pit boundaries. The study reveals a distinct machinery in which two closely associated ROP pathways oppositely regulate cell wall deposition patterns for the establishment of tiny but highly specialized cell wall domains.

Plant vacuoles are dynamic organelles that play essential roles in regulating growth and development. Two distinct models of vacuole biogenesis have been proposed: separate vacuoles are formed by the fusion of endosomes, or the single interconnected vacuole is derived from the endoplasmic reticulum. These two models are based on studies of two-dimensional (2D) transmission electron microscopy and 3D confocal imaging, respectively. Here, we performed 3D electron tomography at nanometre resolution to illustrate vacuole biogenesis in Arabidopsis root cells. The whole-cell electron tomography analysis first identified unique small vacuoles (SVs; 400-1,000 nm in diameter) as nascent vacuoles in early developmental cortical cells. These SVs contained intraluminal vesicles and were mainly derived/matured from multivesicular body (MVB) fusion. The whole-cell vacuole models and statistical analysis on wild-type root cells of different vacuole developmental stages demonstrated that central vacuoles were derived from MVB-to-SV transition and subsequent fusions of SVs. Further electron tomography analysis on mutants defective in MVB formation/maturation or vacuole fusion demonstrated that central vacuole formation required functional MVBs and membrane fusion machineries.

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (T H 1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

A haustorium is the unique organ that invades host tissues and establishes vascular connections. Haustorium formation is a key event in parasitism, but its underlying molecular basis is largely unknown. Here, we use Phtheirospermum japonicum, a facultative root parasite in the Orobanchaceae, as a model parasitic plant. We performed a forward genetic screen to identify mutants with altered haustorial morphologies. The development of the haustorium in P. japonicum is induced by host-derived compounds such as 2,6-dimethoxy-p-benzoquinone. After receiving the signal, the parasite root starts to swell to develop a haustorium, and haustorial hairs proliferate to densely cover the haustorium surface. We isolated mutants that show defects in haustorial hair formation and named them haustorial hair defective (hhd) mutants. The hhd mutants are also defective in root hair formation, indicating that haustorial hair formation is controlled by the root hair development program. The internal structures of the haustoria in the hhd mutants are similar to those of the wild type, indicating that the haustorial hairs are not essential for host invasion. However, all the hhd mutants form fewer haustoria than the wild type upon infection of the host roots. The number of haustoria is restored when the host and parasite roots are forced to grow closely together, suggesting that the haustorial hairs play a role in stabilizing the host-parasite association. Thus, our study provides genetic evidence for the regulation and function of haustorial hairs in the parasitic plant., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Brachypodium distachyon is an emerging model plant for studying biological phenomena in temperate grasses. Study of the growth scale is essential to analyse spatio-temporal changes in molecular factors throughout the life cycle. For sensitive and robust staging based on morphology in B. distachyon, we demonstrated the utility of the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemical industry) scale, which is comparable to the Zadoks scale conventionally used for Triticeae crops. We compared the chronological progression of B. distachyon accessions Bd21 and Bd3-1, in addition to the progression of Chinese Spring wheat. The comparison of growth stages illustrates the morphological similarities and differences in the timing of life cycle events. Furthermore, we compared metabolite accumulation patterns across different growth stages and across different stress conditions using a widely targeted metabolome analysis. Metabolic profiling determined commonalities and specificities in chemical properties that were dependent on organisms, growth stages and/or stress conditions. Most metabolites accumulated equivalently in B. distachyon and wheat. This qualitative similarity indicated the superiority of B. distachyon as a model for Triticeae crops. The growth scale of B. distachyon should provide a conceptual framework for comparative analysis and for knowledge integration between this model grass and crops in the Pooideae subfamily., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

Peroxisomes are essential organelles that are characterized by the possession of enzymes that produce hydrogen peroxide (H2O2) as part of their normal catalytic cycle. During the metabolic process, peroxisomal proteins are inevitably damaged by H2O2 and the integrity of the peroxisomes is impaired. Here, we show that autophagy, an intracellular process for vacuolar degradation, selectively degrades dysfunctional peroxisomes. Marked accumulation of peroxisomes was observed in the leaves but not roots of autophagy-related (ATG)-knockout Arabidopsis thaliana mutants. The peroxisomes in leaf cells contained markedly increased levels of catalase in an insoluble and inactive aggregate form. The chemically inducible complementation system in ATG5-knockout Arabidopsis provided the evidence that these accumulated peroxisomes were delivered to vacuoles for degradation by autophagy. Interestingly, autophagosomal membrane structures specifically recognized the abnormal peroxisomes at the site of the aggregates. Thus, autophagy is essential for the quality control of peroxisomes in leaves and for proper plant development under natural growth conditions.

In micrographs acquired with a transmission electron microscope, 3-dimensional (3D) objects are superimposed onto a 2D screen. This reduction in dimension necessarily leads to a degradation of image resolution. To overcome this problem, 3D microscopy techniques, such as tomography and single particle analysis, have been developed. Tomography has been used to visualize cells in 3D, and single particle analysis has been used to investigate macromolecules and viral particles. In this chapter we will describe how we have collected tilting series micrographs from plant cells and how we have reconstructed the cellular volumes using dual axis electron tomography.

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical-basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical-basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.

Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.