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Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction.
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Transporte Activo de Núcleo Celular/inmunología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Proteínas de la Membrana/metabolismo , Poro Nuclear/inmunología , Transducción de Señal , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Conformación ProteicaRESUMEN
Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.
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The root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated 'The people behind the papers' interview.
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Arabidopsis , Arabidopsis/metabolismo , Autofagia , Separación Celular , Humanos , Orgánulos , Cápsula de Raíz de Planta , Raíces de Plantas/metabolismoRESUMEN
The root cap secretes mucilage and sheds border cells (border-like cells, BLCs) in Arabidopsis (Arabidopsis thaliana). These mucilage and root cap-derived cells form a defensive barrier against soil pathogens. BEARSKIN1 (BRN1) and BRN2 are 2 homologous NAM, ATAF1/2, and CUC2 (NAC) family transcription factors of Arabidopsis, and mucilage secretion is inhibited in the brn1/2 double mutant. BRN1 and BRN2 are also involved in the expression of a pectin-digesting enzyme, POLYGALACTURONASE (RCPG), that facilitates BLC shedding. To further explore the connection between mucilage secretion and BLC shedding, we examined mucilage production in Arabidopsis lines displaying altered BLC detachment. Inactivation of BRN2 blocked mucilage synthesis and secretion, while inactivation of BRN1 and RCPG did not. Interestingly, RCPG sorted into mucilage-carrying vesicles budding from the Golgi and inhibited mucilage secretion in brn2-delayed BLC detachment. The root cap of a germinating seedling is initially covered with a cuticle, which is replaced by mucilage from BLCs as the seedling begins to shed these cells. Ectopic expression of RCPG in germinating seedlings caused early BLC formation and accelerated the cuticle-to-mucilage transition, indicating that RCPG expression and mucilage secretion are co-regulated. Furthermore, brn2 roots exhibited slower growth and increased cell death when subjected to salt or osmotic stress. Our research suggests that BRN2-mediated mucilage secretion contributes to BLC release to build an extracellular defense zone surrounding the root cap.
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The para-crystalline structures of prolamellar bodies (PLBs) and light-induced etioplast-to-chloroplast transformation have been investigated via electron microscopy. However, such studies suffer from chemical fixation artifacts and limited volumes of 3D reconstruction. Here, we examined Arabidopsis thaliana cotyledon cells by electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed scanning transmission ET to image large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice-like carbon atoms in diamonds. Within 2 h after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets (pre-granal thylakoids), which folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids contained curved membranes in their tips, we examined the expression and localization of CURT1 (CURVATURE THYLAKOID1) proteins. CURT1A transcripts were most abundant in de-etiolating cotyledon samples, and CURT1A was concentrated at the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. In contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to cubic crystal growth in the dark.
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Arabidopsis , Tilacoides , Arabidopsis/genética , Arabidopsis/metabolismo , Carbono/metabolismo , Cloroplastos/metabolismo , Cotiledón , Diamante/análisis , Diamante/metabolismo , Tomografía con Microscopio Electrónico , Tilacoides/metabolismo , Zinc/metabolismoRESUMEN
In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.
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Membrana Celular/metabolismo , Pared Celular/metabolismo , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Plantas/metabolismo , Orgánulos/metabolismo , Células Vegetales/metabolismoRESUMEN
The nuclear basket (NB) is an essential structure of the nuclear pore complex (NPC) and serves as a dynamic and multifunctional platform that participates in various critical nuclear processes, including cargo transport, molecular docking, and gene expression regulation. However, the underlying molecular mechanisms are not completely understood, particularly in plants. Here, we identified a guanylate-binding protein (GBP)-like GTPase (GBPL3) as a novel NPC basket component in Arabidopsis. Using fluorescence and immunoelectron microscopy, we found that GBPL3 localizes to the nuclear rim and is enriched in the nuclear pore. Proximity labeling proteomics and protein-protein interaction assays revealed that GBPL3 is predominantly distributed at the NPC basket, where it physically associates with NB nucleoporins and recruits chromatin remodelers, transcription apparatus and regulators, and the RNA splicing and processing machinery, suggesting a conserved function of the NB in transcription regulation as reported in yeasts and animals. Moreover, we found that GBPL3 physically interacts with the nucleoskeleton via disordered coiled-coil regions. Simultaneous loss of GBPL3 and one of the 4 Arabidopsis nucleoskeleton genes CRWNs led to distinct development- and stress-related phenotypes, ranging from seedling lethality to lesion development, and aberrant transcription of stress-related genes. Our results indicate that GBPL3 is a bona fide component of the plant NPC and physically and functionally connects the NB with the nucleoskeleton, which is required for the coordination of gene expression during plant development and stress responses.
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Arabidopsis , Poro Nuclear , Animales , Poro Nuclear/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Matriz Nuclear , Plantas/metabolismoRESUMEN
Nanomaterial-based yarns have been actively developed owing to their advantageous features, namely, high surface-area-to-volume ratios, flexibility, and unusual material characteristics such as anisotropy in electrical/thermal conductivity. The superior properties of the nanomaterials can be directly imparted and scaled-up to macro-sized structures. However, most nanomaterial-based yarns have thus far, been fabricated with only organic materials such as polymers, graphene, and carbon nanotubes. This paper presents a novel fabrication method for fully inorganic nanoribbon yarn, expanding its applicability by bundling highly aligned and suspended nanoribbons made from various inorganic materials (e.g., Au, Pd, Ni, Al, Pt, WO3, SnO2, NiO, In2O3, and CuO). The process involves depositing the target inorganic material on a nanoline mold, followed by suspension through plasma etching of the nanoline mold, and twisting using a custom-built yarning machine. Nanoribbon yarn structures of various functional inorganic materials are utilized for chemical sensors (Pd-based H2 and metal oxides (MOx)-based green gas sensors) and green energy transducers (water splitting electrodes/triboelectric nanogenerators). This method is expected to provide a comprehensive fabrication strategy for versatile inorganic nanomaterials-based yarns.
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Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1LBD) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A-I. Recombinant DTH1LBD bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1LBD in Chlamydomonas phenocopied the dth1 mutant's defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.
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Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Secuencia de Aminoácidos , Metabolismo de los Lípidos/fisiología , Nitrógeno/metabolismo , Fosfolípidos/metabolismo , Fotosíntesis/fisiología , Triglicéridos/metabolismoRESUMEN
Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid ß-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid ß-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.
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Chlamydomonas reinhardtii , Chlamydomonas , Chlamydomonas reinhardtii/metabolismo , Lipasa/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Triglicéridos/metabolismoRESUMEN
Chloroplast development is a complex process that is critical to the growth and development of plants. However, the detailed mechanism of chloroplast development in woody plants remains unclear. In this study, we showed that chloroplasts with elaborate thylakoids could develop from proplastids in the cells of calli derived from leaf tissues of Populus tomentosa upon exposure to light. Chloroplast development was confirmed at the molecular and cellular levels. Transcriptome analysis revealed that genes related to photoreceptors and photosynthesis were significantly up-regulated during chloroplast development in a time-dependent manner. In light-induced chloroplast development, a key process was the removal of hydrogen peroxide, in which thylakoid-localized PtotAPX played a major role; light-induced chloroplast development was enhanced in PtotAPX-overexpressing transgenic P. tomentosa callus with lower levels of hydrogen peroxide, but was suppressed in PtotAPX antisense transgenic callus with higher levels of hydrogen peroxide. Moreover, the suppression of light-induced chloroplast development in PtotAPX antisense transgenic callus was relieved by the exogenous reactive oxygen species scavenging agent N,N'-dimethylthiourea (DMTU). Based on these results, we propose that PtotAPX-mediated removal of reactive oxygen species plays a key role in chloroplast development from proplastids upon exposure to light in P. tomentosa.
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Populus , Tilacoides , Ascorbato Peroxidasas/genética , Cloroplastos , Peróxido de Hidrógeno , Populus/genéticaRESUMEN
Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular prothylakoids (24 h after imbibition [HAI]), sheet-like pregranal thylakoids that develop from the prothylakoids (36 HAI), proliferation of pro-grana stacks with wide tubular connections to the originating pregrana thylakoids (60 HAI), structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI), and conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pregranal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II complex subunits at 36 HAI. ATP synthase, cytochrome b6f, and light-harvesting complex II proteins are detected at 60 HAI, while PSI proteins and the curvature-inducing CURT1A protein appear at 84 HAI. If stromal ribosome biogenesis is delayed, prothylakoids accumulate until stromal ribosomes are produced, and grana-forming thylakoids develop after polysomes bind to the thylakoid membranes. In fzo-like (fzl) mutants, in which thylakoid organization is perturbed, pro-grana stacks in cotyledons form discrete, spiral membrane compartments instead of organelle-wide membrane networks, suggesting that FZL is involved in fusing membrane compartments together. Our data demonstrate that the assembly of thylakoid protein complexes, CURT1 proteins, and FZL proteins mediate distinct and critical steps in thylakoid biogenesis.
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Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cotiledón/microbiología , Dinaminas/metabolismo , Polirribosomas/metabolismo , Tilacoides/metabolismoRESUMEN
The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.
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Imagenología Tridimensional , Membranas Intracelulares/metabolismo , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Vacuolas/metabolismo , Cucumovirus/fisiología , Cucumovirus/ultraestructura , Tomografía con Microscopio Electrónico , Membranas Intracelulares/ultraestructura , Epidermis de la Planta/citología , Epidermis de la Planta/ultraestructura , Epidermis de la Planta/virología , Virus de Plantas/ultraestructura , Fracciones Subcelulares/metabolismo , Nicotiana/citología , Tombusviridae/fisiología , Tombusviridae/ultraestructura , Vacuolas/ultraestructura , Proteínas Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
Chromatin is the main carrier of genetic information and is non-randomly distributed within the nucleus. Next-generation sequence-based chromatin conformation capture technologies have enabled us to directly examine its three-dimensional organization at an unprecedented scale and resolution. In the best-studied mammalian models, chromatin folding can be broken down into three hierarchical levels, compartment, domains, and loops, which play important roles in transcriptional regulation. Although similar structures have now been identified in plants, they might not possess exactly the same functions as the mammalian ones. Here, we review recent Hi-C studies in plants, compare plant chromatin structures with their mammalian counterparts, and discuss the differences between plants with different genome sizes.
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Núcleo Celular , Cromatina , Animales , Regulación de la Expresión Génica , Plantas/genéticaRESUMEN
During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Citocinesis/genética , Citocinesis/fisiología , Dinaminas/genética , Dinaminas/metabolismo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Red trans-Golgi/metabolismo , Red trans-Golgi/fisiologíaRESUMEN
Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Autofagia/fisiología , Proteínas Portadoras/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
BACKGROUND: Estrogen controls the pubertal growth spurt, growth plate closure, and accretion of bone mineral density (BMD) of long bones after biding estrogen receptor (ER). There are two subtypes of ER, ERα and ERß. If each ER subtype has different effects, we may control those actions by manipulating the estrogen binding intensity to each ER subtype and increase the final adult height without markedly reducing BMD or impairing reproductive functions. The purpose of our study was to compare these effects of ERα and ERß on long bones in ovariectomized rats. METHODS: Thirty female rats were ovariectomized and randomly divided into 3 groups. The control, propylpyrazole triol (PPT), and 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) groups were subcutaneously injected for 5 weeks with sesame oil, PPT as an ERα agonist, and DPN as an ERß agonist, respectively. The crown-lump length and body weight were measured weekly. BMD, serum levels of growth hormone (GH) and estradiol were checked before and after 5 weeks of injections. Pituitary GH1 expression levels were determined with quantitative real-time polymerase chain reaction, the proximal tibias were dissected, decalcified and stained with hematoxylin-eosin, and the thicknesses of epiphyseal plates including proliferative and hypertrophic zones were measured in 20-evenly divided sites after 5 weeks of injections. Comparisons for auxological data, serum hormone and pituitary GH1 expression levels, BMD, and epiphyseal plate thicknesses among 3 groups before and after injections were conducted. RESULTS: There was no significant difference in body lengths among 3 groups. The body weights were significantly lower, but, serum GH, pituitary GH1 expression levels, and BMDs were higher in PPT group than the other 2 groups after 5 weeks of injections. There was no significant difference in the thicknesses of the total epiphyseal plate, proliferative, and hypertrophic zone among 3 groups. CONCLUSION: ERα is more involved in pituitary GH secretion and bone mineral deposition than ERß. Weight gain might be prevented with the ERα agonist.
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Tamaño Corporal/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Receptor alfa de Estrógeno/agonistas , Receptor beta de Estrógeno/agonistas , Nitrilos/farmacología , Fenoles/farmacología , Pirazoles/farmacología , Animales , Estradiol/sangre , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/sangre , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Inyecciones Subcutáneas , Nitrilos/administración & dosificación , Nitrilos/química , Ovariectomía , Fenoles/administración & dosificación , Hipófisis/metabolismo , Pirazoles/administración & dosificación , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Structural analyses of organelles and localization of proteins in their confines are essential to investigate inner workings of eukaryotic cells. Electron tomography (ET) is a three-dimensional electron microscopy method with which we can extract sliced views of organelles from any direction and quantify their structural parameters at nanometer-level resolution. This advanced electron microscopy tool is suited for characterization of convoluted membrane compartments and of cellular constituents of dimensions smaller than 100 nm. ET studies of plant cells fixed by rapid freezing have expanded our understanding of the biogenesis and functions of plant organelles. Here we describe how the molecular imaging capacity of correlative light and electron microscopy can be integrated with ET in studies of plant organelles.
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Tomografía con Microscopio Electrónico/métodos , Orgánulos/ultraestructura , Congelación , Imagenología Tridimensional , Células Vegetales/ultraestructura , PresiónRESUMEN
Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.