Comparación de métodos preparativos de tejidos para la extracción de proteínas de la mazorca de cacao (Theobroma cacao L.)

Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B) of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D). Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Methods B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.El cacao, Theobroma cacao L. es uno de los principales cultivos tropicales industriales. La mazorca de cacao tiene un nivel muy alto de sustancias interferentes, tales como polisacáridos y compuestos fenólicos, que podrían impedir el aislamiento adecuado de la proteína. El uso de métodos eficientes de extracción de proteínas es una prioridad para aplicar con éxito los análisis proteómicos. Nosotros comparamos y evaluamos dos métodos preparativos (A y B) de tejidos para la extracción de proteína total mediante el protocolo de extracción con fenol/SDS. La diferencia entre los dos métodos fue extensivos lavados del polvo seco, obtenido mediante trituración con nitrógeno, de la mazorca fueron realizados en el Método A, mientras que un extracto crudo se preparó en el Método B. Extracciones proteicas fueron examinadas utilizando electroforesis monodimensional (1-D). Los resultados muestran que cada método de extracción aisló un único subconjunto del proteoma de las mazorcas de cacao. El análisis de componentes principales mostró poca variación en los datos por el Método A, mientras que el Método B fue poco reproducible, lo que demuestra que el Método A de extracción es un método fiable para la preparación de proteínas de las mazorcas de cacao. Se espera que el protocolo sea aplicable a otros tejidos de plantas recalcitrantes y podría ser de interés para los laboratorios involucrados en análisis de proteómica de plantas. Se recomienda una combinación de los enfoques de extracción para aumentar la cobertura del proteoma utilizando las técnicas de separación a base de gel.This work has been supported by grants from the Senescyt-Government of Ecuador (UTEQ-Ambiental-9-FCAmb-IFOR-2014-FOCICYT002), AMM holds a grant MAEC-AECID (2014-2015) of Spain

Development of MRM methods to quantify proteins isoforms of poliphenol oxidase

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Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), Generalitat de Catalunya (2014SGR215) and European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1-Senescyt-2014) and a grant from UTEQ (UTEQAmbiental-9-FCAmb-IFOR-2014-FOCICYT002)

The role of proteomics in progressing insights into plant secondary metabolism

The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-02; BIO2014-51861-R), European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). JM-C acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1_Senescyt_2014) and a grant from UTEQ (UTEQ-Ambiental-9-FCAmb-IFOR-2014-FOCICYT002)

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Summary Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrolconverting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yield

A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

One of the major intent of metabolic engineering in cell culture systems is to increase yields of secondary metabolites. Efficient transformation methods are a priority to successfully apply metabolic engineering to cell cultures of plants that produce bioactive or therapeutic compounds, such as Vitis vinifera and Taxus x media. The aim of this study was to establish a reliable method to transform non-embryogenic cell cultures of these species. The V. vinifera cv. Gamay/cv. Monastrell cell lines and Taxus x media were used for Agrobacterium-mediated transformation using the Gateway-compatible Agrobacterium sp. binary vector system for fast reliable DNA cloning. The Taxus x media and Vitis cell lines were maintained in culture for more than 4 and 15 months, respectively, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernible effect on cell growth, or led to extracellular accumulation of phytoalexin trans-Resveratrol (t-R) in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA) in the grapevine transgenic cell lines compared to the parental control. The method described herein provides an excellent tool to exploit exponentially growing genomic resources to enhance, optimize or diversify the production of bioactive compounds generated by grapevine and yew cell cultures, and offers a better understanding of many grapevine and yew biology areas.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. holds a postdoctoral grant from SENESCYT-GOVERNMENT OF ECUADOR (006-IECESMG5-GPLR-2012)

Factors Affecting the Bioproduction of Resveratrol by Grapevine Cell Cultures under Elicitation

Here we present a study of the characterization and optimization of the production of trans-Resveratrol (t-R) in grape (Vitis vinifera cv. Gamay) cell cultures elicited with methyl jasmonate (MeJA) and dimethyl-β-cyclodextrin (DIMEB). The aim of this study was to determine the influence of a number of factors of the grapevine cell culture on t-R production level in 250 mL shaken flasks that would enable the better control of this bioproduction system when it is upscaled to a 2 L stirred bioreactor. The factors included the optimal growth phase for elicitation, the concentration of elicitors and of biomass, the order of addition of elicitors, and the illumination regime and ageing of cells. We found out that the optimal biomass density for the production of t-R was 19% (w/v) with an optimal ratio of 0.5 g DIMEB/g biomass. The most productive concentrations of the elicitors tested were 50 mM DIMEB and 100 µM MeJA, reaching maximum values of 4.18 mg·mL−1 and 16.3 mg·g biomass−1 of t-R concentration and specific production, respectively. We found that the order of elicitor addition matters since, as compared with the simultaneous addition of both elicitors, the addition of MeJA 48 h before DIMEB results in ca. 40% less t-R production, whilst there is no significant difference when MeJA is added 48 h after DIMEB. Upon upscaling, the better conditions tested for t-R production were aeration at 1.7 vol/vol/min without agitation, 24 °C, and 30 g·L−1 sucrose, achieving production rates similar to those obtained in shaken flasks.This work has been supported by Spanish Ministry of Science and Innovation, MCIN/AEI/10.13039/501100011033, ERDF A way of making Europe and European Union NextGenerationEU/PRTR grants PID2020-113438RB-I00 and TED2021-129617B-I00, Valencian Conselleria d’Innovació, Universitats, Ciencia y Societat Digital grant CIAICO/2021/167

Comunicación intercultural para el desarrollo de relaciones interpersonales en las comunidades montubias del cantón Quevedo y zonas de influencia

The main objective of the study is to establish the impact of intercultural communication for the development of interpersonal relationships in the Montubia communities of the Quevedo canton and areas of influence, specifically, it was sought to identify the themes of social interest in the dissemination of cultural wealth of Montubia communities, analyze the need for outreach programs and projects that respond to an intercultural dialogue as a reality in permanent construction, where the elements belonging to the various minority cultures serve to enrich knowledge, but not as an obstacle or cultural distancing. The methodology used starts from the inductive method to obtain reliable information on the experiential expressions of the social actors, framed in the interpretive paradigm; Through the application of a structured survey to 269 beneficiaries of the project of social work in the area of ​​Communication, the main results validated by direct observation show the ignorance of the beneficiaries in the communities intervened with social interaction projects on intercultural communication and Montubia culture. It is concluded that it is necessary to promote community programs and projects on intercultural communication that favor the preservation of indigenous cultural customs and traditions, promote intercultural values ​​in educational processes and promote the development of cultural intelligence in changing times.El estudio tiene como objetivo principal establecer el impacto de la comunicación intercultural para el desarrollo de relaciones interpersonales en las comunidades montubias del cantón Quevedo y zonas de influencia, de manera específica se buscó, identificar las temáticas de interés social en la difusión de las riquezas culturales de las comunidades montubias, analizar la necesidad de programas y proyectos de vinculación que respondan a un diálogo intercultural como una realidad en permanente construcción, donde los elementos pertenecientes a las diversas culturas minoritarias sirvan para el enriquecimiento del conocimiento, más no como un obstáculo o distanciamiento cultural. La metodología utilizada parte desde el método inductivo para obtener una información fidedigna sobre las expresiones vivenciales de los actores sociales, enmarcado en el paradigma interpretativo; mediante la aplicación de una encuesta estructurada a 269 beneficiarios del proyecto de labor social en área de Comunicación. Los principales resultados validados mediante observación directa, demuestra el desconocimiento de los beneficiarios en las comunidades intervenidas con proyectos de interacción social sobre la comunicación intercultural y cultura montubia. Se concluye que es necesario promover programas y proyectos comunitarios sobre comunicación intercultural que favorezca la preservación de las costumbres y tradiciones autóctonas culturales, fomentar valores interculturales en los procesos educativos e impulsar el desarrollo la inteligencia cultural en épocas cambiantes

Prenylated Flavonoids of the Moraceae Family: A Comprehensive Review of Their Biological Activities

Prenylated flavonoids (PFs) are natural flavonoids with a prenylated side chain attached to the flavonoid skeleton. They have great potential for biological activities such as anti-diabetic,anti-cancer, antimicrobial, antioxidant, anti-inflammatory, enzyme inhibition, and anti-Alzheimer’s effects. Medicinal chemists have recently paid increasing attention to PFs, which have become vital for developing new therapeutic agents. PFs have quickly developed through isolation and semi- or full synthesis, proving their high value in medicinal chemistry research. This review comprehensively summarizes the research progress of PFs, including natural PFs from the Moraceae family and their pharmacological activities. This information provides a basis for the selective design and optimization of multifunctional PF derivatives to treat multifactorial diseases.This work has been supported by the Spanish Ministry of Science and Innovation, MCIN/AEI/10.13039/501100011033, ERDF A way of making Europe, and European Union NextGenerationEU/PRTR grants PID2020-113438RB-I00 and TED2021-129617B-I00, and Valencian Conselleria d’Innovació, Universitats, Ciencia y Societat Digital grant CIAICO/2021/167. This work was also supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contract No. 451-03-66/2024-03/200007)

Phosphoprotein enrichment from soluble and microsomal fractions of grapevine (vitis vinifera cv. gamay) cell culture using metal oxide affinity chromatography (MOAC)

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