Miras Moreno, Begoña

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Miras Moreno, Begoña

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Biología Vegetal

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Open Access
Estudio metabolómico y genómico de cultivos celulares de zanahoria
(2018-01-09) Miras Moreno, Begoña; Almagro Romero, Lorena; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Facultad de Biología
El objetivo principal de este trabajo de investigación fue caracterizar los metabolitos secundarios producidos en dos líneas celulares de zanahoria. Para ello, uno de los objetivos concretos fue la caracterización de la producción de compuestos de naturaleza isoprenoide y compuestos fenólicos en suspensiones celulares de una línea celular verde de Dacus carota en condiciones control y en condiciones de elicitación con ciclodextrinas, jasmonato de metilo, hexenol y β-glucano separadamente o en combinación. De la misma manera, se caracterizó la producción de isoprenoides y compuestos fenólicos en suspensiones celulares de una línea celular naranja de D. carota en condiciones control y en condiciones de elicitación con ciclodextrinas y jasmonato de metilo. Asimismo, se estudio la producción de fitosteroles y carotenoides y la expresión de genes implicados en su ruta biosintética en suspensiones celulares de una línea celular naranja de D. carota en presencia de inhibidores de la ruta de biosíntesis de carotenoides y fitosteroles. Los resultados derivados de este estudio muestran que la línea celular de zanahoria verde biosintetizó de manera constitutiva los siguientes isoprenoides: carotenoides, α-tocoferol, clorofilas a y b y fitosteroles. El principal isoprenoide encontrado en la línea verde fue la luteína, una xantofila amarilla. La caracterización de la línea celular de zanahoria verde mostró que los fitoesteroles y compuestos fenólicos se acumularon principalmente en el medio extracelular (15100 μg/L y 477,5 μg/L, respectivamente) en presencia de ciclodextrinas. A diferencia de los compuestos mencionados anteriormente, el β-caroteno (1138,1 μg/L), la luteína (25949,5 μg/L), el α-tocoferol (8063,8 μg/L) y la clorofila a (1625,1 μg/L) y b (9958,3 μg/L) se acumularon principalmente dentro de las células. Por lo tanto, los ciclodextrinas fueron capaces de inducir la ruta biosintética del mevalonato, aumentando la biosíntesis de fitoesteroles y compuestos fenólicos, y acumulándolos fuera de las células. Sin embargo, en ausencia de ciclodextrinas, las células de la zanahoria acumularon principalmente carotenoides a través de la ruta biosintética del 4-fosfato de metileritritol. Por lo tanto, el uso de ciclodextrinas permitiría la acumulación extracelular de fitoesteroles y compuestos fenólicos incrementando el flujo de carbono hacia la ruta citosólica del mevalonato y hacia la ruta de biosíntesis fenilpropanoide. Además, β-glucano fue capaz de inducir la acumulación intracelular de α-tocoferol en la línea de células de zanahoria verde. Por otro lado, la línea celular de zanahoria naranja biosintetizó de manera constitutiva carotenoides y fitoesteroles. Los principales isoprenoides acumulados en esta línea celular fueron β-caroteno y luteína. El análisis de esta línea naranja en condiciones de elicitación (50 mM ciclodextrinas) mostró que los fitoesteroles se acumulaban principalmente en el medio extracelular. Sin embargo, el β-caroteno y la luteína se acumularon esencialmente dentro de las células. El tratamiento combinado de ciclodextrinas y jasmonato de metilo mejoró la acumulación extracelular de compuestos fenólicos. Por lo tanto, la línea celular de zanahoria naranja bajo condición de elicitación proporciona un sistema biotecnológico eficiente para producir compuestos bioactivos. La adición de Terbinafina y Diflufenican que inhiben las rutas biosintéticas de fitoesteroles y carotenoides respectivamente, provocó una alta acumulación de escualeno y fitoeno. Estos compuestos bioactivos no suelen acumularse y, debido a esto, la línea celular de zanahoria naranja tratada con estos inhibidores es una fuente alternativa para producirlos. Los niveles de expresión más altos del gen escualeno sintasa se encontraron después de 24h de tratamiento con el inhibidor terbinafina. Esto puede explicar la alta acumulación intracelular de escualeno sin efecto negativo sobre los niveles de fitosteroles. Además, las ciclodextrinas no aumentaron los niveles de expresión de escualeno sintasa. Este hecho puso de manifiesto que los ciclodextrinas no funcionan como moléculas inductoras en la biosíntesis de los fitoesteroles, sino que son capaces de extraerlos de las membranas celulares. Por último, los niveles de expresión del gen fitoeno desaturasa 1 sugirieron que la acumulación del contenido total de carotenoides podría estar vinculada a la expresión de este gen. Sin embargo, el tratamiento con diflufenican disminuyó los niveles de expresión de fitoeno sintasa 1 y fitoeno desaturasa, lo que sugiere que la alta acumulación de fitoeno no se correlacionó con un alto nivel de expresión de estos genes. The main objective was to characterize secondary metabolites produced in two carrot cell lines. Thus, one of the specific objectives was to characterize the production of isoprenoid and phenolic compounds in cell suspensions of a Daucus carota green cell line under control conditions and under elicitation conditions with cyclodextrins, methyl jasmonate, Hexenol and β-glucan separately or in combination. Moreover, the production of isoprenoids and phenolic compounds was characterized in cell suspensions of orange D. carota cell line under control conditions and under conditions of elicitation with cyclodextrins and methyl jasmonate. It was also studied the production of phytosterols and carotenoids and the expression of genes involved in their biosynthetic pathway in cell suspensions of orange D. carota cell line in the presence of inhibitors of the biosynthesis pathway of carotenoids and phytosterols. The results derived from this study refer to the green carrot cell line constitutively biosynthesized the following isoprenoids: carotenoids, α-tocopherol, chlorophyll a and b, and phytosterols. The main isoprenoid found in this green carrot line was lutein, a yellow xanthophyll. The characterization of the green carrot cell line showed that phytosterols and phenolic compounds wew mainly accumulated in the extracellular medium (15100 μg/L and 477.5 μg/L, respectively) in the presence of cyclodextrins. Unlike the aforementioned compounds, β-carotene (1138.1 μg/L), lutein (25949.5 μg/L), α-tocopherol (8063.8 μg/L) and chlorophyll 1625.1 μg/L) and b (9958.3 μg / L) were mainly accumulated within the cells. Therefore, cyclodextrins were able to induce the biosynthetic pathway of mevalonate, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulating them outside the cells. However, in the absence of cyclodextrins, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate biosynthetic pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of phytosterols and phenolic compounds by increasing the flow of carbon towards the mevalonate cytosolic pathway and towards the phenylpropanoid pathway. In addition, β-glucan was able to induce the intracellular accumulation of α-tocopherol in the green carrot cell line. On the other hand, the orange carrot cell line constitutively biosynthesized carotenoids and phytosterols. The major isoprenoids accumulated in this cell line were β-carotene and lutein. Analysis of this orange line under elicitation conditions (50 mM cyclodextrins) showed that phytosterols accumulated mainly in the extracellular medium. However, β-carotene and lutein were essentially accumulated within the cells. Combined treatment of cyclodextrins and methyl jasmonate improved the extracellular accumulation of phenolic compounds. Therefore, the orange carrot cell line under elicitation condition provides an efficient biotechnological system to produce bioactive compounds. The addition of Terbinafine and diflufenican, which inhibited the biosynthetic pathways of phytosterols and carotenoids respectively, caused a high accumulation of squalene and phytoene. These bioactive compounds are not usually accumulated and, because of this, the orange carrot cell line treated with these inhibitors is an alternative source to produce them. The highest expression levels of the square gene were found after 24h of treatment with the inhibitor terbinafine. This fact may explain the high intracellular accumulation of squalene with no negative effect on phytosterol levels. In addition, the cyclodextrins did not increase the expression levels of squalene synthase. This fact revealed that cyclodextrins do not function as induction molecules in the biosynthesis of phytosterols, but are capable of extracting them from cell membranes. Finally, gene expression levels of squalene synthase suggest that the accumulation of total carotene content could be linked to the expression of this gene. However, treatment with diflufenican decreased expression levels of phytoene synthase 1 and phytoene desaturase, suggesting that high phytoene accumulation did not correlate with a high level of expression of these genes.
Open Access
Cyclodextrins increase phytosterol and tocopherol levels in suspension cultured cells obtained from mung beans and safflower
(Wiley, 2017-07-13) Tudela, Libertad Raquel; Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Biología Vegetal
In this work, suspension-cultured cells of mung beans and safflower were used in order to analyze the effect of methyl jasmonate and/or cyclodextrins, on bioactive compound production such as phytosterols and tocopherols. The results indicated that mung bean suspension-cultured cells produced higher amount of total phytosterols and tocopherols. In particular, mung bean suspension-cultured cells produced almost 220-fold higher levels of tocopherols than safflower suspension-cultured cells in the best conditions. However, while cyclodextrins were able to enhance extracellular production of phytosterols, in the case of tocopherols, they only increased their intracellular accumulation. Our results showed that mung bean cells could be used as a highly efficient system for the production of phytosterols and tocopherols which have a wide range of biological activities.
Open Access
Neurofuzzy logic predicts a fine-tuning metabolic reprogramming on elicited Bryophyllum PCSCs guided by salicylic acid
(Frontiers Media, 2022-09-23) Lozano-Milo, Eva; Zhang, Leilei; Landin, Mariana; Lucini, Luigi; Gallego, Pedro P.; Miras Moreno, Begoña; García Pérez, Pascual
Novel approaches to the characterization of medicinal plants as biofactories have lately increased in the field of biotechnology. In this work, a multifaceted approach based on plant tissue culture, metabolomics, and machine learning was applied to decipher and further characterize the biosynthesis of phenolic compounds by eliciting cell suspension cultures from medicinal plants belonging to the Bryophyllum subgenus. The application of untargeted metabolomics provided a total of 460 phenolic compounds. The biosynthesis of 164 of them was significantly modulated by elicitation. The application of neurofuzzy logic as a machine learning tool allowed for deciphering the critical factors involved in the response to elicitation, predicting their influence and interactions on plant cell growth and the biosynthesis of several polyphenols subfamilies. The results indicate that salicylic acid plays a definitive genotype-dependent role in the elicitation of Bryophyllum cell cultures, while methyl jasmonate was revealed as a secondary factor. The knowledge provided by this approach opens a wide perspective on the research of medicinal plants and facilitates their biotechnological exploitation as biofactories in the food, cosmetic and pharmaceutical fields.
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Enhanced accumulation of phytosterols and phenolic compounds incyclodextrin-elicited cell suspension culture of Daucus carota
(Elsevier, 2016-06-15) Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Biología Vegetal
Inthis work, suspension-culturedcells of Daucus carota wereusedto evaluate the effect of -cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100 g L−1 and 477.46 g L−1, respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, -carotene (1138.03 g L−1), lutein (25949.54 g L−1) and -tocopherol (8063.82 g L−1) chlorophyll a (1625.13 g L−1) and b (9.958 (9958.33 g L−1) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols andphenolic compounds bydiverting the carbonflux towards the cytosolicmevalonate/phenylpropanoid pathway.
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The metabolomics reveals intraspecies variability of bioactive compounds in elicited suspension cell cultures of three Bryophyllum species
(Elsevier, 2021-02-15) García-Pérez, Pascual; Lucini, Luigi; Gallego, Pedro P.; Miras Moreno, Begoña; García Pérez, Pascual
In this work, the combination of elicited plant suspension cultured cells (PSCCs) with untargeted metabolomics establishes a powerful, cutting-edge strategy to unravel the effects of elicitors on the biosynthetic potential of medicinal plants, as Bryophyllum sp. The PSCC technology constitutes a successful biotechnological system for the study and production of bioactive compounds throughout the inclusion of elicitors with the ability to modulate secondary metabolism. The use of methyl jasmonate (MJ) and salicylic acid (SA) as abiotic elicitors on bryophyllum PSCCs, resulted in differential effects on cell growth and secondary metabolism, depending on the species, including synergistic and antagonistic effects. This fact suggests that both elicitors play a pleiotropic effect on plant secondary metabolism, showing complex interactions, according to the UHPLC-QTOF mass spectrometry profiling. Thus, the combination of both elicitors induced a strong synergistic on B. daigremontianum PSCCs, with 2272 putatively annotated compounds, whereas it caused a negative effect on the secondary metabolism of B. × houghtonii PSCCs, being MJ the only elicitor driving a positive effect, presenting 2972 annotated compounds. Meanwhile, B. tubiflorum PSCCs did not show a significant modulation of secondary metabolism, with 1521 annotated compounds. The metabolite annotation indicated that three families of secondary metabolites were mainly affected by elicitation: phenolic compounds constituted the most affected family by elicitation, mainly represented by flavonoids and lignans; N-containing compounds included glucosinolates, amines, and alkaloids, reported to Bryophyllum sp. for the first time; and terpenoids included mainly phytoalexins and saponins. The results depict a deep genotype-dependent metabolomic reprogramming of secondary metabolism in response to elicitors, thanks to the application of untargeted metabolomics. This knowledge will allow the consideration of Bryophyllum sp. as a valuable source of bioactive compounds, with the potential associated to PSCCs, for being included in food, cosmetic, and pharmaceutical applications.
Open Access
Effect of diflufenican on total carotenoid and phytoene production in carrot suspension‑cultured cells
(Springer, 2018-08-06) Fraser, Paul D.; Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Biología Vegetal
This work analyzes the efect of difufenican, an inhibitor of phytoene desaturase, on the gene expression profles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10 µM difufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7 days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10 µM difufenican after 7 days of incubation in the presence of light and with 30 g/L sucrose in the culture medium. Moreover, difufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This difufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of difufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.
Open Access
Bioactivity and bioavailability of phytoene and strategies to improve its production
(SPRINGER, 2018-12-12) Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Biología Vegetal
Embargo
Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells
(Springer, 2018-04-21) Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Biología Vegetal
Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight−1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.
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Bioactivity of phytosterols and their production in plant in vitro cultures
(American Chemical Society, 2016-09-12) Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Sabater Jara, Ana Belén; Biología Vegetal
Phytosterols are a kind of plant metabolite belonging to the triterpene family. These compounds are essential biomolecules for human health, and so they must be taken from foods. β-Sitosterol, campesterol, and stigmasterol are the main phytosterols found in plants. Phytosterols have beneficial effects on human health since they are able to reduce plasma cholesterol levels and have antiinflammatory, antidiabetic, and anticancer activities. However, there are many difficulties in obtaining them, since the levels of these compounds produced from plant raw materials are low and their chemical synthesis is not economically profitable for commercial exploitation. A biotechnological alternative for their production is the use of plant cell and hairy root cultures. This review is focused on the biosynthesis of phytosterols and their function in both plants and humans as well as the different biotechnological strategies to increase phytosterol biosynthesis. Special attention is given to describing new methodologies based on the use of recombinant DNA technology to increase the levels of phytosterols.
Open Access
Accumulation and tolerance of cadmium in a nonmetallicolous ecotype of Silene vulgaris Garcke (Moench)
(Murcia: Servicio de Publicaciones de la Universidad de Murcia, 2014) Ferrer, María Angeles; Almagro Romero, Lorena; Miras Moreno, Begoña; Pedreño García, María Ángeles; Biología Vegetal
En este estudio, se analizó el efecto de diferentes concentraciones de Cd2+ sobre un ecotipo de Silene vulgaris Garcke (Moench). La concentración de 60 μM de Cd2+ provocó una ligera inhibición del crecimiento de las plantas mientras que la concentración más alta (120 μM) redujo drásticamente la biomasa y la elongación de la raíz y los brotes. Además, se detectaron altos niveles de Cd2+ en las plantas, un coeficiente de bioacumulación elevado en las raices y un bajo factor de translocación indicando que el ecotipo de S. vulgaris empleado en este estudio presenta una alta capacidad de acumulación de Cd2+ en las raíces y sería un buen candidato para la fitoestabilización, lo que contribuiría a reducir los niveles de Cd2+ en el suelo. Además, los resultados obtenidos indican que se debe tener precaución con el origen de esta planta, ya que podría representar una fuente adicional de Cd2+ en la dieta humana.