RESUMEN
Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue-light-dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue-light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop-and-go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma-membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2- and photosynthesis-dependent signals. The present study might add novel regulatory mechanism for light-dependent plant organelle interactions.
Asunto(s)
Arabidopsis/metabolismo , Células del Mesófilo/metabolismo , Mitocondrias/metabolismo , Fotosíntesis/fisiología , Fototropinas/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cloroplastos/fisiología , Células del Mesófilo/fisiología , Microscopía Fluorescente , Mitocondrias/fisiología , Fotosíntesis/genéticaRESUMEN
Circumnutation is a plant growth movement in which the tips of axial organs draw a circular orbit. Although it has been studied since the nineteenth century, its mechanism and significance are still unclear. Greened adzuki bean (Vigna angularis) epicotyls exhibited a clockwise circumnutation in the top view with a constant period of 60 min under continuous white light. The bending zone of circumnutation on the epicotyls was always located in the region 1-3 cm below the tip, and its basal end was almost identical to the apical end of the region where the epicotyl had completely elongated. Therefore, epidermal cells that construct the bending zone are constantly turning over with their elongation growth. Since exogenously applied auxin transport inhibitors and indole-3-acetic acid (IAA) impaired circumnutation without any effect on the elongation rate of epicotyls, we attempted to identify the distribution pattern of endogenous auxin. Taking advantage of its large size, we separated the bending zone of epicotyls into two halves along the longitudinal axis, either convex/concave pairs in the plane of curvature of circumnutation or pre-convex/pre-concave pairs perpendicular to the plane. By liquid chromatography-mass spectrometry, we found, for the first time, that IAA and gibberellin A1 were asymmetrically distributed in the pre-convex part in the region 1-2 cm below the tip. This region of epicotyl sections exhibited the highest responsiveness to exogenously applied hormones, and the latent period between the hormone application and the detection of a significant enhancement in elongation was 15 min. Our results suggest that circumnutation in adzuki bean epicotyls with a 60 min period is maintained by differential growth in the bending zone, which reflects the hormonal status 15 min before and which is shifting sequentially in a circumferential direction. Cortical microtubules do not seem to be involved in this regulation.
Asunto(s)
Giberelinas/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Vigna/metabolismo , Cromatografía Liquida , Espectrometría de Masas , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Vigna/crecimiento & desarrolloRESUMEN
A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure.
Asunto(s)
Arabidopsis/fisiología , Citoesqueleto de Actina/efectos de la radiación , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Muerte Celular/efectos de la radiación , Núcleo Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Luz , Células del Mesófilo/citología , Células del Mesófilo/fisiología , Células del Mesófilo/efectos de la radiación , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiaciónRESUMEN
In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria.
Asunto(s)
Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Hydrocharitaceae/citología , Hydrocharitaceae/efectos de la radiación , Luz , Células Vegetales/metabolismo , Epidermis de la Planta/citología , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Reacciones Cruzadas , Genes de Plantas , Hydrocharitaceae/genética , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Péptidos/metabolismo , Fosforilación/efectos de la radiación , Fototropinas/química , Fototropinas/metabolismo , Células Vegetales/efectos de la radiación , Epidermis de la Planta/efectos de la radiación , Alineación de SecuenciaRESUMEN
The morphology of plant nuclei varies among different species, organs, tissues and cell types. However, mechanisms and factors involved in the maintenance of nuclear morphology are poorly understood. Because nuclei retain their shapes even after cytoskeletal inhibitor treatments both in vivo and in vitro, we assumed involvement of the nuclear lamina, which plays a critical role in the regulation of nuclear morphology in animals. The crude nuclear lamina fraction isolated from Arabidopsis thaliana leaves was analyzed by mass spectrometry, and putative nuclear lamina proteins were identified. Among their T-DNA insertion lines, nuclei of little nuclei1 (linc1) and linc4 disruptants were more spherical than those of wild-type plants. Because A. thaliana harbors four LINC genes, we prepared all single and linc1/4 and linc2/3 double disruptants. In leaf epidermal cells, the circularity index of the nucleus in all linc disruptants except linc3 was significantly higher than that in the wild-type plants. The extent of the effects of LINC1 and/or LINC4 disruption was significantly higher than that of the effects of LINC2 disruption. The nuclear area was significantly smaller in the linc1, linc4 and linc1/4 disruptants than in the wild-type plants. Regardless of the defects in nuclear morphology, all linc disruptants exhibited a normal ploidy level. In interphase cells, LINC1 and LINC4 were mainly localized to the nuclear periphery, whereas LINC2 was in the nucleoplasm and LINC3 was detected in both regions. From prometaphase to anaphase in mitotic root tip cells, LINC1 was co-localized with chromosomes, whereas other LINCs were dispersed in the cytoplasm.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citoplasma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismoRESUMEN
Hepatocyte nuclear factor (HNF) 4alpha is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4alpha. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3'-untranslated region (3'-UTR), and those for miR-34a (MRE34a) were identified in the 3'-UTR in HNF4alpha mRNA. The HNF4alpha protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4alpha mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3'-UTR of HNF4alpha was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4alpha coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3'-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4alpha by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4alpha was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.
Asunto(s)
Ciclo Celular/fisiología , Factor Nuclear 4 del Hepatocito/metabolismo , MicroARNs/fisiología , Regiones no Traducidas 3'/genética , Western Blotting , Ciclo Celular/genética , Electroforesis en Gel de Poliacrilamida , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Humanos , MicroARNs/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chloroplasts are actively anchored at the appropriate intracellular regions to maintain advantageous distribution patterns under specific environmental conditions. Redistribution of chloroplasts is accompanied by their de-anchoring and re-anchoring, respectively, from and to the cortical cytoplasm. In spinach mesophyll cells, high-intensity blue light and Ca(2+) treatment induced the disappearance of the meshwork-like array of actin filaments surrounding chloroplasts, which was suppressed by a calmodulin antagonist. Regulatory mechanisms of chloroplast anchoring were investigated using plasma membrane (PM) ghosts, on which the cortical cytoplasm underlying the PM was exposed. Addition of an actin-depolymerizing reagent or > 1 µM Ca(2+) induced detachment of a substantial number of chloroplasts from the PM ghosts concomitant with disordered actin organization. Calmodulin antagonists and anti-calmodulin antibodies negated the effects of Ca(2+). In addition, Ca(2+)-induced detachment of chloroplasts was no longer evident on the calmodulin-depleted PM ghosts. We propose that chloroplasts are anchored onto the cortical cytoplasm through interaction with the actin cytoskeleton, and that Ca(2+)-calmodulin-sensitized de-anchoring of chloroplasts is a critical early step in chloroplast redistribution induced by environmental stimuli.
Asunto(s)
Actinas/fisiología , Calcio/fisiología , Calmodulina/fisiología , Cloroplastos/fisiología , Células del Mesófilo/fisiología , Spinacia oleracea/citología , Citoesqueleto de Actina/fisiología , Citoplasma/fisiología , Luz , Spinacia oleracea/fisiologíaRESUMEN
In epidermal and mesophyll cells of Arabidopsis (Arabidopsis thaliana) leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue light receptor phototropin2 in mesophyll cells. Here, we investigate the involvement of phototropin and the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness, whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in Arabidopsis, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Arabidopsis/crecimiento & desarrollo , Fototropinas/metabolismo , Hojas de la Planta/citología , Arabidopsis/genética , Benzamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Luz , Tiazolidinas/farmacologíaRESUMEN
PURPOSE: Peroxisome proliferator-activated receptor α (PPARα) is an important transcriptional factor that regulates genes encoding endo/xenobiotic enzymes and lipid metabolizing enzymes. In this study, we investigated whether microRNAs (miRNAs) are involved in the regulation of PPARα in human liver. METHODS: Precursor or antisense oligonucleotide for miR-21 or miR-27b was transfected into HuH7 cells; expression of PPARα and acyl-CoA synthetase M2B was determined by Western blot and real-time RT-PCR. Luciferase assay was performed to identify the functional miRNA recognition element (MRE). Expression levels of PPARα, miR-21, and miR-27b in a panel of 24 human livers were determined. RESULTS: The overexpression and inhibition of miR-21 or miR-27b in HuH7 cells significantly decreased and increased the PPARα protein level, respectively, but not PPARα mRNA level. The miRNA-dependent regulation of PPARα affected the expression of its downstream gene. Luciferase assay identified a functional MRE for miR-21 in the 3'-untranslated region of PPARα. In human livers, the PPARα protein levels were not correlated with PPARα mRNA, but inversely correlated with the miR-21 levels, suggesting a substantial impact of miR-21, although the contribution of miR-27b could not be ruled out. CONCLUSIONS: We found that PPARα in human liver is regulated by miRNAs.
Asunto(s)
Regulación de la Expresión Génica , Hígado/fisiología , MicroARNs/genética , PPAR alfa/genética , Regiones no Traducidas 3' , Línea Celular Transformada , Línea Celular Tumoral , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Hígado/metabolismo , MicroARNs/metabolismo , PPAR alfa/metabolismo , ARN Mensajero/genética , TransfecciónRESUMEN
In mesophyll cells of the aquatic monocot Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.
RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Cobre/metabolismo , Lámina Nuclear , Proteínas Nucleares , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Hibridación Fluorescente in Situ , Mutación/genética , Lámina Nuclear/genética , Lámina Nuclear/metabolismo , Lámina Nuclear/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA-SeqRESUMEN
Human vitamin D3 hydroxylase (CYP24) catalyzes the inactivation of 1alpha,25-dihydroxyvitamin D3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3'-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P < 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.
Asunto(s)
Calcitriol/antagonistas & inhibidores , MicroARNs/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Esteroide Hidroxilasas/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Biocatálisis , Western Blotting , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Calcitriol/farmacología , Línea Celular Tumoral , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunohistoquímica , ARN Mensajero/genética , Esteroide Hidroxilasas/genética , Vitamina D3 24-HidroxilasaRESUMEN
Most of the biological effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are elicited by the binding to vitamin D receptor (VDR), which regulates gene expression. Earlier studies reported no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through translational repression or mRNA degradation. A potential miR-125b recognition element (MRE125b) was identified in the 3'-untranslated region of human VDR mRNA. We investigated whether VDR is regulated by miR-125b. In luciferase assays using a plasmid containing the MRE125b, the antisense oligonucleotide for miR-125b significantly increased (130% of control) the reporter activity in KGN cells, whereas the precursor for miR-125b significantly decreased (40% of control) the reporter activity in MCF-7 cells, suggesting that miR-125b functionally recognized the MRE125b. By electrophoretic mobility shift assays, it was demonstrated that the overexpression of miR-125b significantly decreased the endogenous VDR protein level in MCF-7 cells to 40% of control. 1,25(OH)(2)D(3) drastically induced the CYP24 mRNA level in MCF-7 cells, but the induction was markedly attenuated by the overexpression of miR-125b. In addition, the antiproliferative effects of 1,25(OH)(2)D(3) in MCF-7 cells were significantly abolished by the overexpression of miR-125b. These results suggest that the endogenous VDR level was repressed by miR-125b. In conclusion, we found that miR-125b posttranscriptionally regulated human VDR. Since the miR-125b level is known to be downregulated in cancer, such a decrease may result in the upregulation of VDR in cancer and augmentation of the antitumor effects of 1,25(OH)(2)D(3).
Asunto(s)
Regiones no Traducidas 3'/genética , Regulación Neoplásica de la Expresión Génica/fisiología , MicroARNs/fisiología , Neoplasias/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Proliferación Celular/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética , Humanos , Luciferasas/metabolismo , Neoplasias/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.
Asunto(s)
Arabidopsis/efectos de la radiación , Luz , Mitocondrias/efectos de la radiación , Hojas de la Planta/ultraestructura , Arabidopsis/citología , Proteínas Fluorescentes Verdes/química , Microscopía Fluorescente , Mitocondrias/metabolismo , Hojas de la Planta/efectos de la radiación , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/efectos de la radiaciónRESUMEN
The intracellular distribution of organelles plays a pivotal role in the maintenance and adaptation of a wide spectrum of cellular activities in plants. Chloroplasts are a special type of organelle able to photosynthesize, capturing light energy to fix atmospheric CO2. Consequently, the intracellular positioning of chloroplasts is crucial for plant growth and development. Knowledge of the photoreceptors and cellular apparatus responsible for chloroplast movement has gradually accumulated over time, yet recent advances have allowed improved understanding. In this article, several aspects of research progress into the mechanisms for maintaining the specific intracellular distribution patterns of chloroplasts, namely, chloroplast anchoring, are summarized, together with a brief consideration of the future prospects of this subject. Our discussion covers developmental, physiological, ecophysiological, and recent cell biological research areas.
Asunto(s)
Cloroplastos/fisiología , Fenómenos Fisiológicos de las Plantas , Cloroplastos/efectos de la radiación , Fotosíntesis , Desarrollo de la Planta , Plantas/efectos de la radiaciónRESUMEN
Petiole curvature is important in regulating light interception by the leaf. To dissect the determination processes of leaf angle, we irradiated the lamina or petiole of Chenopodium album L. with either one or two spots of actinic light, after dark adaptation. When the abaxial side of the petiole was irradiated with blue light, the petiole curvature increased, and under continuous irradiation, the curvature continued for up to 6 h. The rate of curvature increased with increasing blue light intensity. The curvature induced by irradiation of the abaxial side with blue light ceased when the adaxial side of the petiole was simultaneously irradiated with either blue or red light. When an inhibitor for photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, was applied to the adaxial side of the petiole, the cessation of curvature caused by blue light was only weakly inhibited, while the cessation caused by red light was markedly inhibited. When the adaxial side of the petiole was irradiated alternately with red and far-red light, the far-red light antagonized the cessation of curvature caused by the red light. These results clearly show that the petiole curvature is controlled by two processes, the induction and the cessation of curvature. At least three photoreceptor systems, blue-light receptor, photosynthesis and phytochrome, are involved in the reactions.
Asunto(s)
Chenopodium album/fisiología , Hojas de la Planta/fisiología , Chenopodium album/efectos de los fármacos , Chenopodium album/efectos de la radiación , Diurona/farmacología , Luz , Células Fotorreceptoras/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/efectos de la radiaciónRESUMEN
MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression through translational repression or mRNA cleavage. Here, we found that cytochrome P450 (CYP), a superfamily of drug-metabolizing enzymes, is a target of miRNA. Human CYP1B1, which is highly expressed in estrogen target tissues, catalyzes the metabolic activation of various procarcinogens and the 4-hydroxylation of 17beta-estradiol. CYP1B1 protein is abundant in cancerous tissues. We identified a near-perfect matching sequence with miR-27b in the 3'-untranslated region of human CYP1B1. Luciferase assays revealed that the reporter activity of the plasmid containing the miR-27b recognition element was decreased in MCF-7 cells (miR-27 positive) but not in Jurkat cells (miR-27b negative). Exogenously expressed miR-27b could decrease the luciferase activity in Jurkat cells. In MCF-7 cells, the antisense oligoribonucleotide for miR-27b restored the luciferase activity and increased the protein level and enzymatic activity of endogenous CYP1B1. These results suggested that human CYP1B1 is post-transcriptionally regulated by miR-27b. The expression levels of miR-27b and CYP1B1 protein in breast cancerous and adjacent noncancerous tissues from 24 patients were evaluated. In most patients, the expression level of miR-27b was decreased in cancerous tissues, accompanied by a high level of CYP1B1 protein. A significant inverse association was observed between the expression levels of miR-27b and CYP1B1 protein. Thus, the decreased expression of miR-27b would be one of causes of the high expression of CYP1B1 protein in cancerous tissues. This is the first study to show that miRNAs regulate not only essential genes for physiologic events but also drug-metabolizing enzymes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , MicroARNs/genética , Regiones no Traducidas 3' , Adulto , Anciano , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Citocromo P-450 CYP1B1 , Femenino , Células HeLa , Humanos , Células Jurkat , Luciferasas/biosíntesis , Luciferasas/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Chloroplasts are known to maintain specific intracellular distribution patterns under specific environmental conditions, enabling the optimal performance of photosynthesis. To this end, chloroplasts are anchored in the cortical cytoplasm. In leaf epidermal cells of aquatic monocot Vallisneria, we recently demonstrated that the anchored chloroplasts are rapidly de-anchored upon irradiation with high-intensity blue light and that the process is probably mediated by the blue-light receptor phototropins. Chloroplast de-anchoring is a necessary step rendering the previously anchored chloroplasts mobile to allow their migration. In this article, based on the results obtained in Vallisneria together with those in other plant species, we briefly discussed possible modes of regulation of chloroplast anchoring and de-anchoring by actin cytoskeleton. The topics include roles of photoreceptor systems, actin-filament-dependent and -independent chloroplast anchoring, and independence of chloroplast de-anchoring from actomyosin and microtubule systems.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Cloroplastos/metabolismo , Actinas/metabolismo , Luz , Microtúbulos/metabolismo , Fotosíntesis/fisiologíaRESUMEN
PURPOSE: Some blood biomarkers or histological examination by liver biopsy are used for the diagnosis of liver diseases in clinics. However, conventional blood biomarkers show poor specificity and sensitivity, and liver biopsy is highly invasiveness. Therefore, to overcome such disadvantages, specific/sensitive and noninvasive options are desirable. In recent years, circulating microRNAs (miRNAs) have been acknowledged for their potential as disease markers. Actually, several miRNAs have been reported to be biomarker candidates of liver diseases. However, these earlier studies were performed for one disease. Therefore, the specificity as biomarkers was not guaranteed, because they didn't study for the other types of liver injury. In this study, we examined if circulating miRNA could distinguish different types of liver diseases. METHODS: Serum miRNA profiles in 28 patients with chronic hepatitis B, chronic hepatitis C, primary biliary cirrhosis, autoimmune hepatitis, nonalcoholic steatohepatitis or drug-induced liver injury as well as 4 control subjects were determined by TaqMan MicroRNA Array analysis. Principal component analysis (PCA) of selected miRNAs was performed. RESULTS: We identified 37 miRNAs whose levels were significantly different between any of the groups. Although individual miRNAs could not distinguish different types of liver diseases, probably because of similar liver pathology, their profiling by PCA could classify different liver disease groups. CONCLUSIONS: The profiling of the selected miRNAs can be useful to distinguish different types of liver diseases.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Hepatitis B Crónica/genética , Hepatitis C Crónica/genética , Hepatitis Autoinmune/genética , Cirrosis Hepática Biliar/genética , MicroARNs/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Adulto , Anciano , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Femenino , Hepatitis B Crónica/sangre , Hepatitis C Crónica/sangre , Hepatitis Autoinmune/sangre , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática Biliar/sangre , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangreRESUMEN
Cytochrome P450 (CYP) 1B1, which catalyzes 17beta-estradiol 4-hydroxylation, is expressed in steroid-related tissues including ovary, testis, and adrenal gland. Generally, the expressions of steroidogenic CYPs are transcriptionally regulated by steroidogenic factor-1 (SF-1) and cAMP response element (CRE) binding protein (CREB). In the present study, we examined the possibility that the human CYP1B1 gene might be regulated by SF-1 and CREB. Gel shift analyses revealed that in vitro translated SF-1 can bind to the putative SF-1 binding sites, SF-1a (at -1722) and SF-1b (at -2474), on the CYP1B1 gene. In vitro translated CREB barely binds to the putative SF-1 binding sites. Luciferase analysis revealed that a reporter plasmid, pGL3 (-2623/+25), containing the SF-1a and SF-1b elements is transactivated by the concomitant co-expression of SF-1 and protein kinase A (PKA). However, the transcriptional activity is induced by PKA alone. Mutations in the SF-1a and SF-1b elements did not affect the luciferase activity. Thus, the binding of SF-1 to the putative SF-1 binding sites of the human CYP1B1 gene might not be essential for transcriptional regulation. Interestingly, deletion and mutation analyses indicated that the PKA signaling pathway is involved in the xenobiotic responsive element (XRE)-mediated transactivation of the human CYP1B1 gene.