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Fisiología comparada de una línea celular clorofílica y una mutante amarilla del zacate Bouteloua gracilis (Kunth) Lag ex Griffiths en condiciones de estrés osmótico

BETZAIDA JIMENEZ FRANCISCO (2013)

Tesis (Maestría en Ciencias, especialista en Botánica).- Colegio de Postgraduados, 2013.

Las suspensiones celulares clorofílicas de plantas superiores son una herramienta valiosa para estudiar las respuestas y señales a estrés hídrico asociadas con el cloroplasto. Debido a su importancia metabólica los cloroplastos actúan como sensores de los cambios ambientales y de las redes complejas de señales que coordinan las actividades celulares y ayudan a la célula durante el estrés. En el presente trabajo se realizó la caracterización fisiológica de dos líneas celulares en suspensión del zacate B. gracilis, que se caracterizan, una por ser altamente clorofílica y, la otra, una mutante deficiente de clorofila; se estudió el efecto de la deficiencia de clorofila en la respuesta al estrés osmótico mediante la evaluación de las relaciones hídricas, crecimiento, cambios en los niveles de pigmentos clorofílicos y carotenoides y en la acumulación de prolina y ácido abscísico (ABA). Y el efecto de un periodo de exposición a 0.375 µM de ABA en las variables antes mencionadas. La deficiencia de clorofila en la línea mutante estuvo relacionada con daños en la estructura del cloroplasto, concentraciones bajas de carotenoides y menor acumulación de biomasa fresca. El estrés osmótico provocó disminución en el potencial hídrico y osmótico con efectos negativos en la acumulación de peso fresco para las células clorofílicas. Mientras que el peso seco solo se vio afectado en las células mutantes. Otros resultados encontrados fueron el incremento en la concentración de clorofila, carotenoides y ABA con el incremento de la tensión osmótica en el medio. Las células mutantes no mostraron cambios significativos en el contenido de clorofila. Contrariamente los niveles de carotenoides, prolina y ABA si presentaron incremento en estas células, aunque en menor grado a los observados en la línea clorofílica. La exposición a ABA previamente a la aplicación del estrés osmótico no mostró efecto en cuanto al comportamiento en las variables mencionadas anteriormente en comparación con el tratamiento en el cual las células no fueron expuestas al ABA. Los resultados obtenidos proporcionan información relevante para la realización de estudios posteriores sobre la participación del cloroplasto en la respuesta de la célula vegetal a estrés osmótico. _______________ COMPARATIVE PHYSIOLOGY OF A CHLOROPHYLLIC AND A YELLOW MUTANT CELL LINE OF Bouteloua gracilis (Kunth) Lag. ex Griffiths IN CONDITIONS OF OSMOTIC STRESS. ABSTRACT: Chlorophyllous cell suspensions from higher plants are a valuable tool for studying the responses to water stress and signal associated with the chloroplast. Because of its metabolic importance chloroplasts act as sensors of environmental changes and complex signaling networks that coordinate cellular activities and help the cell during stress. In this work, it was carried out the physiological characterization of two cell suspension lines of the grass B. gracilis, one of them characterized by being highly chlorophyllous and the other one for being a mutant deficient in chlorophyll accumulation is presented. We also studied the effect of the chlorophyll deficiency in the cell response to osmotic stress by evaluating water relations, growth, changes in the levels of chlorophyll and carotenoid pigments, as well as the accumulation of proline and abscisic acid (ABA). ABA is involved in major routes of adaptation to environmental stresses, so we studied the effect of cell exposure to 0.375 uM of this growth regulator in the aforementioned variables. The chlorophyll deficiency in the mutant line was associated with damage to the chloroplast structure, low concentrations of carotenoids and lower accumulation of fresh biomass. The osmotic stress induced a decrease in water and osmotic potential with negative effects on the accumulation of fresh weight for chlorophyllous cells, while the dry weight was only affected in mutant cells. Other results were the increase in the concentration of chlorophyll, carotenoids and ABA with increasing osmotic tension in the medium. Mutant cells showed no significant change in the content of chlorophyll; on the contrary carotenoid, proline and ABA levels in these cells did show an increase, however, they were low with respect to the chlorophyll cell line. Exposure of cells to ABA as a pre-stress treatment did not affect the studied variables in comparison to cells directly grown in the hyperosmotic medium. The results obtained provide relevant information for further studies on the chloroplas involvement of the plant cell response to osmotic stress.

Master thesis

Estrés hídrico ABA Prolina Biogénesis de cloroplasto Biosíntesis de clorofila Pigmentos fotosintéticos Drought stress Proline Chloroplast biogenesis Chlorophyll biosynthesis Photosynthetic pigments Botánica Maestría CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA

Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2

ISRAEL MARURI LOPEZ JUAN FRANCISCO JIMENEZ BREMONT (2017)

"The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other."

Article

BiFC Heterodimer Homodimer Chloroplast localization Polyamines BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR

Fibrillarin from Archaea to human

ULISES RODRIGUEZ CORONA MARGARITA SOBOL LUIS CARLOS RODRIGUEZ ZAPATA PAVEL HOZAK ENRIQUE CASTAÑO DE LA SERNA (2015)

Fibrillarin is an essential protein that is well known as a molecular marker of transcriptionally active RNA polymerase I. Fibrillarin methyltransferase activity is the primary known source of methylation for more than 100 methylated sites involved in the first steps of preribosomal processing and required for structural ribosome stability. High expression levels of fibrillarin have been observed in several types of cancer cells, particularly when p53 levels are reduced, because p53 is a direct negative regulator of fibrillarin transcription. Here, we show fibrillarin domain conservation, structure and interacting molecules in different cellular processes as well as with several viral proteins during virus infection.

Article

CANCER METHYLATION P53 RIBOSOMAL BIOGÉNESIS RNA PROCESSING BIOLOGÍA Y QUÍMICA BIOLOGÍA Y QUÍMICA

Fibrillarin from Archaea to human

ULISES RODRIGUEZ CORONA MARGARITA SOBOL LUIS CARLOS RODRIGUEZ ZAPATA PAVEL HOZAK ENRIQUE CASTAÑO DE LA SERNA (2015)

Fibrillarin is an essential protein that is well known as a molecular marker of transcriptionally active RNA polymerase I. Fibrillarin methyltransferase activity is the primary known source of methylation for more than 100 methylated sites involved in the first steps of preribosomal processing and required for structural ribosome stability. High expression levels of fibrillarin have been observed in several types of cancer cells, particularly when p53 levels are reduced, because p53 is a direct negative regulator of fibrillarin transcription. Here, we show fibrillarin domain conservation, structure and interacting molecules in different cellular processes as well as with several viral proteins during virus infection.

Article

CANCER METHYLATION P53 RIBOSOMAL BIOGÉNESIS RNA PROCESSING BIOLOGÍA Y QUÍMICA

Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2

ISRAEL MARURI LOPEZ JUAN FRANCISCO JIMENEZ BREMONT (2017)

"The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other."

Article

BiFC Heterodimer Homodimer Chloroplast localization Polyamines BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR

Characterization of chloroplast region rrn16-rrn23S from the tropical timber tree Cedrela odorata L. and de novo construction of a transplastomic expression vector suitable for Meliaceae trees and other economically important crops

LUISA ALHUCEMA LOPEZ OCHOA MAX MIZRAIM APOLINAR HERNANDEZ YURI JORGE JESUS PEÑA RAMIREZ (2015)

The forest tree Spanish cedar (Cedrela odorata L.) is well-known for its high-value timber; however, this species is attacked by the shoot borer (Hypsipyla grandella) during its early years of development, resulting in branched stems and making the plants useless for high-quality wood production. The generation of resistant varieties expressing entomotoxic proteins may be an alternative to pesticide treatments. The use of plastid transformation rather than nuclear transformation should be used because it reduces the risk of transgene dissemination by pollen. Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. Thus, C. odorata plastome sequences are a prerequisite. The rrn16-rrn23 plastome region was selected, cloned, and characterized. When the sequence identity among the rrn16-rrn23 regions from C. odorata and Nicotiana tabacum was compared, 3 inDels of 240, 104, and 39 bp were found that might severely affect transformation efficiency. Using this region, a new transformation vector was developed using pUC19 as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from C. odorata and adding 2 independent expression cassettes into the trnI-trnA intergenic region, conferring spectinomycin resistance, the ability to express the gfp reporter gene, and a site that can be used to express any other gene of interest.

Article

CHLOROPLAST TRANSFORMATION EXPRESSION VECTOR FLANKING REGIONS PLASTOME SEQUENCE TROPICAL TREE BIOTECHNOLOGY BIOLOGÍA Y QUÍMICA BIOLOGÍA Y QUÍMICA

Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2

Israel Maruri López JUAN FRANCISCO JIMENEZ BREMONT (2017)

"The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other."

Article

BiFC Heterodimer Homodimer Chloroplast localization Polyamines BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR

Papel del receptor a estrógenos GPER en los efectos de la (-)-epicatequina sobre la biogénesis mitocondrial en células C2C12

Role of estrogen receptor GPER in the effects of (-)-Epicatechin on mitochondrial biogenesis in C2C12 myotubes

ADRIANA MIRANDA CERVANTES (2017)

Se ha evidenciado la capacidad de (-)-epicatequina ((-)-EPI) de estimular la biogénesis mitocondrial (BiM) en el músculo esquelético (ME) en ratones. Sin embargo, los mecanismos por los cuales (-)-EPI promueve dichos efectos permanecen desconocidos. Previamente se ha identificado el papel de un receptor a estrógenos acoplado a proteínas G (GPER) en la modulación de los efectos cardiovasculares de (-)-EPI. Por lo tanto, la hipótesis de este trabajo es que el GPER media (al menos parcialmente) los efectos estimulatorios de (-)-EPI sobre la BiM en células de ME. Para comprobar la hipótesis se utilizó un modelo in vitro de células de mioblastos de ME de ratón (C2C12), los cuales se diferenciaron a miotubos. Mediante microscopia confocal se identificó la presencia del GPER en la superficie celular y el citoplasma en miotubos C2C12. Los miotubos se trataron con (-)-EPI a distintas concentraciones (3-10 μM) y se obtuvo como resultado la estimulación de la BiM, incremento la membrana interna mitocondrial (MitoTracker Green FM, tinción de fluorescencia), incremento en la membrana externa (niveles de la proteína porina), aumento en factores de transcripción involucrados en la estimulación de la BiM (Factor de respiración nuclear 2 NRF-2, factor de transcripción mitocondrial A , TFAM) , así como los niveles de actividad de la enzima citrato sintasa. Sumado a estos efectos se observó que los miotubos tratados con (-)-EPI mostraron mayor longitud y ancho comparado con el grupo control. Los efectos de (-)-EPI sobre la mitocondria de los miotubos y el tamaño celular fueron mayores que aquéllos observados con el agonista del GPER G-1. El uso de un antagonista selectivo de GPER, G-36, evidencio un bloqueo parcial de los parámetros medidos tras la estimulación con (-)-EPI o G-1. De tal manera estos resultados indican que el GPER se expresa en células musculares y media al menos parcialmente los efectos estimulatorios de (-)-EPI sobre la BiM. Por lo tanto, la activación de GPER puede contribuir a los efectos estimulatorios de (-)-EPI E sobre la estructura y función del ME.

Skeletal muscle (SkM) represents ~40% of the total body mass and plays an important role in locomotion, thermogenesis and energy balance. Aging/disease-associated sarcopenia is the progressive loss of SkM strength and mass linked to a decrease in SkM mitochondrial function and biogenesis (MB, synthesis of new mitochondria). Sarcopenia is associated with lower quality of life and increased mortality and there is no specific pharmacological treatment. Thus, it is crucial to find new molecules to treat the disease. (-)Epicatechin ((-)-EPI), a natural cacao flavanol, has been shown to increase SkM strength and function in animals and humans; these effects are associated to the modulation of SkM mitochondrial function. However, the exact mechanism by which (-)-EPI exerts its effects on SkM remains unknown. The G-protein coupled estrogen receptor (GPER) binds and signals via 17-b estradiol, an estrogen that is structurally similar to (-)-EPI. The aim of this work was to evaluate the role of GPER in mediating (-)-EPI effects on MB and function using C2C12 myotubes as a model of SkM. Methods: GPER presence in myotubes was evaluated by confocal microscopy. Cells were treated with increasing concentrations of (-)-EPI (1-10 µM) for 48 h. We evaluated MB by mitochondrial staining with MitoTracker® Green and quantification of protein levels of complex II, IV and nuclear respiratory factor 2 (NRF-2). Mitochondrial function was studied by quantification of citrate synthase and MTT activity. Statistical analysis was performed using one-way ANOVA or Student´s t-test, as appropriate. Results: data indicate that GPER is localized at the cell membrane and cytoplasm and that 10 μM (-)-EPI significantly increased MB (20-50 %) and function endpoints (15-20 %) compared to non-treated cells; effects were blocked by G36 (GPER antagonist). Conclusion: evidence suggests that (-)-EPI effects on SkM mitochondria are mediated by GPER and highlight this receptor as a potential therapeutic candidate for the treatment of aging/disease-associated sarcopenia.

Master thesis

Músculo esquelético (ME), (-)-epicatequina (-)-EPI, biogénesis mitocondrial, GPER, sarcopenia Skeletal muscle, mitochondrial biogenesis, (-)-epicatechin, GPER, sarcopenia BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA MOLECULAR BIOLOGÍA MOLECULAR