Estudio del efecto del aumento de la temperatura asociado a las condiciones del cambio climático sobre el olivo (Olea europaea L.), mediante el uso integrado de técnicas de fisiología y bioquímica y bioquímica clásica, proteómica, metabolómica y transcriptómica

This Doctoral Thesis is focused on olive tree (Olea europaea L. subsp. europaea), the dominant tree crop over large areas of the Western Mediterranean Basin, with a production of 20,872,788 tonnes in 2018. Olive oil is appreciated worldwide because of its chemical composition, nutraceutical value, and organoleptic properties. Like most crops, the olive tree is facing important problems and challenges related to adverse environmental conditions, either biotic or abiotic ones, a situation that can be worsened in a climate change scenario. Hence, understanding the physiological and molecular basis/mechanism of olive tree adaptability and resilience to different environmental stresses, especially to high temperature, is crucial for predicting the consequences of climate warming, for implementing agricultural practices, designing classic or biotechnological breeding programs, and, lately, increasing its production in order to cope with the current demand. In the present work, both the effect and the responses to an increase of 4 °C above ambient temperature have been studied at phenological, physiological and molecular,-omics, levels, in an attempt to integrate all the results in the novel Systems Biology direction. The use of the –omics techniques (transcriptomics, proteomics, and metabolomics), and its integration with physiological and phenological studies constitute the great novelty of this work. It has required an important effort in optimizing different protocols for transcriptome (RNA isolation and qRT-PCR), proteome (shotgun nLC-MS/MS Orbitrap), and metabolome (UHPLC-MS/MS Q-tof) profiling, together with computational tools for the identification and quantification of the different biomolecules, their functional classification, statistical analysis, clustering and networking. Both newly developed tools and knowledge generated will establish the basis for ongoing and future studies to obtain a better understanding of the molecular mechanisms mediating phenotypes of interest (productive, tolerant to environmental cues, nutraceutical value and the selectionof genotypes more resilient to climate warming). The manuscript has been organized and presented in nine chapters as follows: Chapter I summarises the state of the art of the topics covered in this thesis: the experimental system, objectives, hypothesis and methodologies. It starts with the description of the olive tree as a plant species and crop, its economic and cultural importance, problems and challenges related to its cultivation. The chapter continues with the description of, and publications on, environmental stresses with an emphasis on warming temperatures and the denominated “Climate Change”. Responses and mechanisms of tolerance to temperature stress and climate warming will be discussed. This chapter finishes with the description of the methodology employed and the integration of the different approaches. Chapter II presents the general and specific objectives of the Thesis. Chapter III corresponds to two published papers on the warming effect on olive tree phenology: “An approach to global warming effects on flowering and fruit set of olive trees growing under field conditions” and “Global warming effects on yield and fruit maturation of olive trees growing under field conditions” Both were published in Scientia Horticulturae. They present alterations in the flowering and ripening processes, caused by the permanent thermal increase of 4oC above ambient temperatura (AT) applied to adult olive trees (cv. `Picual´) growth underfield conditions. In this chapter, three years were surveyed, analysed and data analysed in relation to climate conditions. In general, earlier and prolonged flowering and ripening were observed under warmer (AT+4oC) conditions. Temperature increase reduced the quantity of perfect (well- developed) flowers and fruits. A decrease in oil quality-related chemical parameters (fat, phenolics, and anthocyanins) was observed under thermal increase conditions. Chapter IV corresponds to two published papers on the effect of different temperature regimes in roots and shoots of olive tree seedlings (cv. Arbequina), a plant growth chamber experiment: “Effect of moderate high temperature on the vegetative growth and potassium allocation in olive plants” and “Effects of olive root warming on potassium transport and plant growth” They were published in Journal of Plant Physiology. The effect of differential thermal 37oC /25oC, exposurebetween root and aerial parton growth, and K uptake and transport were evaluated. At 37oC root, 25oC shoot, potassium uptake increased. 37 °C in both organs inhibited growth, reduced K uptake and transport, and diminished water content. Chapters V, VI and VII are devoted to the different –omics approaches (proteomics, transcriptomics and metabolomics) employed in the molecular study of the effect and responses to warming temperature (+ 4oC) in fruit pulp at three ripening stages: green (0), turning red (called veraison) (3) and purple epicarp (4). A discrepancy between the colouring of the skin, traditionally observed as a reference for phenology studies and harvesting, and the olive pulp’s internal ripeness stage and chemical composition has been found. The veraison stage was the one most affected by the temperature increase. Overall, a reduction in the abundance of major compounds related to olive oil quality (lipids, phenylpropanoids, flavonoids, and terpenes) was observed under warming conditions. Chapter VIII contains an integration of all the data, and a general discussion, with focus on those most relevant parameters from an agronomic point of view, those related to crop productivity, either from a quantitative (fruit production) or qualitative (fruit chemical composition) perspective. Based on the present study, a predictive hypothesis on the olive tree situation and production in a future climate change scenario is presented, proposing alternative management practices to deal with the social demand for this crop. Chapter IX corresponds to the conclusions.Esta Tesis Doctoral se centra en el olivo (Olea europaea L. subsp. europaea), el cultivo arbóreo dominante en grandes áreas de la cuenca del Mediterráneo Occidental, con una producción de 20.872.788 toneladas en 2018. El aceite de oliva es apreciado en todo el mundo por su composición química, su valor nutracéutico y sus propiedades organolépticas. Como la mayoría de los cultivos, el olivo se enfrenta a importantes problemas y desafíos relacionados con las condiciones ambientales adversas, ya sean bióticas o abióticas, situación que puede empeorar en un escenario de cambio climático. Por lo tanto, comprender la base/mecanismo fisiológico y molecular de la adaptabilidad y resiliencia del olivo a las diferentes tensiones ambientales, especialmente a altas temperaturas, es crucial para predecir las consecuencias del calentamiento climático, implementar prácticas agrícolas, diseñar programas de mejoramiento genético clásicos o biotecnológicos y, en los últimos tiempos, aumentar su producción a fin de hacer frente a la demanda actual. En el presente trabajo se han estudiado tanto el efecto como las respuestas a un aumento de 4 °C por encima de la temperatura ambiente a nivel fenológico, fisiológico y molecular, -ómico, en un intento de integrar todos los resultados en la nueva dirección de la Biología de Sistemas. El uso de las técnicas de la -ómica (transcriptómica, proteómica y metabolómica) y su integración con estudios fisiológicos y fenológicos constituyen la gran novedad de este trabajo. Se ha solicitado un importante esfuerzo en la optimización de diferentes protocolos de perfiles de transcriptoma (aislamiento de ARN y qRT-PCR), proteoma (Orbitrap de escopeta nLC-MS/MS) y metaboloma (UHPLC-MS/MS Q-tof), junto con herramientas computacionales para la identificación y cuantificación de las diferentes biomoléculas, su clasificación funcional, análisis estadístico, clustering y networking. Tanto las herramientas de nuevo desarrollo como los conocimientos generados sentarán las bases de los estudios en curso y futuros para obtener una mejor comprensión de los mecanismos moleculares que median los fenotipos de interés (productivos, tolerantes a las señales ambientales, de valor nutracéutico y a la selección de genotipos más resistentes al calentamiento climático). El manuscrito ha sido organizado y presentado en nueve capítulos como a continuación se detallan: El capítulo I resume el “estado del arte” de los temas tratados en esta tesis, el sistema experimental, los objetivos, las hipótesis y las metodologías. Comienza con la descripción del olivo, como especie vegetal y cultivo, su importancia económica y cultural, problemas y retos relacionados con su cultivo. El capítulo continúa con la descripción y publicaciones sobre el estrés ambiental con énfasis en el calentamiento de las temperaturas y el denominado "Cambio Climático". Se discutirán las respuestas y los mecanismos de tolerancia al estrés de la temperatura y al calentamiento climático. Este capítulo finaliza con la descripción de la metodología empleada y la integración de los diferentes enfoques. En el capítulo II se presentan los objetivos generales y específicos de la Tesis. El capítulo III corresponde a dos artículos publicados sobre el efecto de calentamiento en la fenología del olivo: "Una aproximación a los efectos del calentamiento global sobre la floración y el cuajado de los olivos que crecen en condiciones de campo". y "Efectos del calentamiento global sobre el rendimiento y la maduración de los frutos del cultivo del olivo en condiciones de campo". Ambos fueron publicados en Scientia Horticulturae. Presenta alteraciones en los procesos de floración y maduración, causadas por un aumento térmico permanente de 4 °C por encima de la temperatura ambiente (AT) aplicada a olivos adultos (cv. `Picual´) en condiciones de campo. En este capítulo se han estudiado, analizado y analizado los datos relativos a las condiciones climáticas durante tres años. En general, se observó una floración y maduración más temprana y prolongada en condiciones más cálidas (AT+4 °C). El aumento de la temperatura redujo la cantidad de flores y frutos perfectos (bien desarrollados). Se observó una disminución de los parámetros químicos relacionados con la calidad del aceite (rendimiento graso, fenólicos y antocianos) en condiciones de aumento térmico. El capítulo IV corresponde a dos artículos publicados sobre el efecto de los diferentes regímenes de temperatura en las raíces y brotes de las plántulas de olivo (cv. `Arbequina´), un experimento de cámara de crecimiento vegetal: "Efecto de las altas temperaturas moderadas sobre el crecimiento vegetativo y la asignación de potasio en las plantas de olivo". y "Efectos del calentamiento de la raíz del olivo en el transporte de potasio y el crecimiento de las plantas" Ambos fueron publicados en Journal of Plant Physiology. Se evaluó el efecto de la diferencia térmica de 25 °C/37 °C, la exposición entre la raíz y la parte aérea sobre el crecimiento, y la absorción y el transporte de K+. En la raíz a 25 °C, brote a 37 °C, la absorción de potasio aumentó. 37 °C en ambos órganos inhibió el crecimiento, redujo la absorción de K+ y el transporte, y disminuyó el contenido de agua. Los capítulos V, VI y VII están dedicados a los diferentes enfoques -ómicos (proteómica, transcriptómica y metabolómica) empleados en el estudio molecular del efecto y las respuestas al calentamiento de la temperatura (+4 °C) en la pulpa del fruto en tres etapas de maduración: verde (0), rojo (llamado envero) (3) y epicarpio púrpura (4). Se ha encontrado una discrepancia entre la coloración de la piel, tradicionalmente observada como referencia para los estudios fenológicos y la recolección, y la fase de maduración interna de la pulpa de la aceituna y la composición química. La fase de envero fue la más afectada por el aumento de temperatura. En general, se observó una reducción en la abundancia de los principales compuestos relacionados con la calidad del aceite de oliva (lípidos, fenilpropanoides, flavonoides y terpenos) en condiciones de calentamiento. El capítulo VIII contiene una integración de todos los datos y una discusión general, centrada en los parámetros más relevantes desde el punto de vista agronómico, relacionados con la productividad de los cultivos, ya sea desde el punto de vista cuantitativo (producción de fruta) o cualitativo (composición química de la aceituna). A partir del presente estudio se presentan hipótesis predictivas sobre la situación del olivo y la producción en un futuro escenario de cambio climático, proponiendo prácticas de gestión alternativas para hacer frente a la demanda social de este cultivo. El capítulo IX corresponde a las conclusiones

A Multi-Omics Analysis Pipeline for the Metabolic Pathway Reconstruction in the Orphan Species Quercus ilex

Holm oak (Quercus ilex) is the most important and representative species of the Mediterranean forest and of the Spanish agrosilvo-pastoral “dehesa” ecosystem. Despite its environmental and economic interest, Holm oak is an orphan species whose biology is very little known, especially at the molecular level. In order to increase the knowledge on the chemical composition and metabolism of this tree species, the employment of a holistic and multi-omics approach, in the Systems Biology direction would be necessary. However, for orphan and recalcitrant plant species, specific analytical and bioinformatics tools have to be developed in order to obtain adequate quality and data-density before to coping with the study of its biology. By using a plant sample consisting of a pool generated by mixing equal amounts of homogenized tissue from acorn embryo, leaves, and roots, protocols for transcriptome (NGS-Illumina), proteome (shotgun LC-MS/MS), and metabolome (GC-MS) studies have been optimized. These analyses resulted in the identification of around 62629 transcripts, 2380 protein species, and 62 metabolites. Data are compared with those reported for model plant species, whose genome has been sequenced and is well annotated, including Arabidopsis, japonica rice, poplar, and eucalyptus. RNA and protein sequencing favored each other, increasing the number and confidence of the proteins identified and correcting erroneous RNA sequences. The integration of the large amount of data reported using bioinformatics tools allows the Holm oak metabolic network to be partially reconstructed: from the 127 metabolic pathways reported in KEGG pathway database, 123 metabolic pathways can be visualized when using the described methodology. They included: carbohydrate and energy metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, and biosynthesis of secondary metabolites. The TCA cycle was the pathway most represented with 5 out of 10 metabolites, 6 out of 8 protein enzymes, and 8 out of 8 enzyme transcripts. On the other hand, gaps, missed pathways, included metabolism of terpenoids and polyketides and lipid metabolism. The multi-omics resource generated in this work will set the basis for ongoing and future studies, bringing the Holm oak closer to model species, to obtain a better understanding of the molecular mechanisms underlying phenotypes of interest (productive, tolerant to environmental cues, nutraceutical value) and to select elite genotypes to be used in restoration and reforestation programs, especially in a future climate change scenario

Responses and Differences in Tolerance to Water Shortage under Climatic Dryness Conditions in Seedlings from Quercus spp. and Andalusian Q. ilex Populations

Analyzing differences in tolerance to drought in Quercus spp., and the characterization of these responses at the species and individual population level, are imperative for the selection of resilient elite genotypes in reforestation programs. The main objective of this work was to evaluate differences in the response and tolerance to water shortage under in five Quercus spp. and five Andalusian Q. ilex populations at the inter- and intraspecies level. Six-month-old seedlings grown in perlite were subjected to drought treatments by withholding water for 28 days under mean 37 °C temperature, 28 W m-2 solar irradiance, and 41% humidity. The use of perlite as the substrate enabled the establishment of severe drought stress with reduction in water availability from 73% (field capacity) to 28% (dryness), corresponding to matric potentials of 0 and −30 kPa. Damage symptoms, mortality rate, leaf water content, photosynthetic, and biochemical parameters (amino acids, sugars, phenolics, and pigments) were determined. At the phenotypic level, based on damage symptoms and mortality, Q. ilex behaved as the most drought tolerant species. Drought caused a significant decrease in leaf fluorescence, photosynthesis rate, and stomatal conductance in all Quercus spp. analyzed, being less pronounced in Q. ilex. There were not differences between irrigated and non-irrigated Q. ilex seedlings in the content of sugar and photosynthetic pigments, while the total amino acid and phenolic content significantly increased under drought conditions. As a response to drought, living Q. ilex seedlings adjust stomata opening and gas exchange, and keep hydrated, photosynthetically active, and metabolically competent. At the population level, based on damage symptoms, mortality, and physiological parameters, the eastern Andalusian populations were more tolerant than the western ones. These observations inform the basis for the selection of resilient genotypes to be used in breeding and reforestation programs

Proteomic analysis of goat milk kefir: profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress

Spatial dynamics of bovine tuberculosis in the Autonomous Community of Madrid, Spain (2010-2012)

Progress in control of bovine tuberculosis (bTB) is often not uniform, usually due to the effect of one or more sometimes unknown epidemiological factors impairing the success of eradication programs. Use of spatial analysis can help to identify clusters of persistence of disease, leading to the identification of these factors thus allowing the implementation of targeted control measures, and may provide some insights of disease transmission, particularly when combined with molecular typing techniques. Here, the spatial dynamics of bTB in a high prevalence region of Spain were assessed during a three year period (2010-2012) using data from the eradication campaigns to detect clusters of positive bTB herds and of those infected with certain Mycobacterium bovis strains (characterized using spoligotyping and VNTR typing). In addition, the within-herd transmission coefficient (β) was estimated in infected herds and its spatial distribution and association with other potential outbreak and herd variables was evaluated. Significant clustering of positive herds was identified in the three years of the study in the same location ("high risk area"). Three spoligotypes (SB0339, SB0121 and SB1142) accounted for >70% of the outbreaks detected in the three years. VNTR subtyping revealed the presence of few but highly prevalent strains within the high risk area, suggesting maintained transmission in the area. The spatial autocorrelation found in the distribution of the estimated within-herd transmission coefficients in herds located within distances <14 km and the results of the spatial regression analysis, support the hypothesis of shared local factors affecting disease transmission in farms located at a close proximity

Sobre moléculas, genes y plantas. Biología molecular del endospermo de los cereales

El presente articulo revisa sucintamente un conjunto de investigaciones sobre la biología molecular de un tejido vegetal, el endospermo de los cereales, que puede considerarse el producto comestible mas importante a escala mundial. en dichas investigaciones se han utilizado técnicas bioquímicas, citogeneticas y de ingeniería genética para el conocimiento básico y la manipulación practica del conjunto de especies cultivadas denominadas cereales. los estudios realizados han abarcado los siguientes aspectos principales: purificación y caracterización de proteínas, caracterización y localizacion cromosomica de genes estructurales y reguladores involucrados en el control genético de la composición bioquímica del tejido, clonaje de genes de plantas en bacterias, y transferencia genica entre especies vegetales. las aplicaciones practicas derivadas hasta ahora incluyen desde métodos para la identificación de especies, variedades, y materias primas, a la transferencia de genes de interés agronómico desde especies silvestres a cultivadas. a medio plazo, los avances realizados en la manipulación in vitro de genes vegetales serán de utilidad practica en la aplicación de la ingeniería genética a la mejora vegetal

Ingeniería molecular en "Triticum"

Se resumen las investigaciones realizadas sobre genética bioquímica de la especie aloploide de Triticum aestivum L. (trigo común). Dichas investigaciones abarcan los siguientes aspectos: a) localización cromosómica de genes que controlan sistemas moleculares; b) formas de interacción entre los genomios integrados en dicha especie; c) transferencia genética extraespecífica y extragenérica desde Aegilops ventricosa, Sécale cereale y Agropyron elongatum a trigo. Se especula sobre las implicaciones evolutivas de los resultados obtenidos y se enumeran las aplicaciones prácticas

PROPUESTA DE INTERVENCIÓN PSICOSOCIAL EN LAS PRÁCTICAS PARENTALES CON ESCOLARES ADOLESCENTES Y SU RELACIÓN CON LA SINTOMATOLOGÍA DEPRESIVA

This work is part of the research, theoretical and practical results of family studies at the Chair for the Study of Integral Training of Students of the University of the East, in collaboration with academic bodies of several universities in Mexico on the Net Research "Family and Human Development". psychosocial intervention in parenting practices of parents of teenagers as a process of social transformation that aims to promote self-management in identifying and solving their needs are considered, emphasizing the need to co-manage the formation of their daughters and children, on the basis of a critical engagement that strengthens the sense of belonging, identity, and the establishment of a social commitment. This proposal will sensitize parents to the care of adolescents in relation to the prevention or the presence of depressive symptoms, through the implementation of a program in which different techniques and procedures and validated by the group are used research.&nbsp; El presente trabajo es parte de los resultados investigativos, teóricos y prácticos de los estudios de familia, en la Cátedra de Estudios de la Formación Integral de los Estudiantes de la Universidad de Oriente, en colaboración con cuerpos académicos de varias universidades de México en la Red de Investigación “Familia y Desarrollo Humano”. Se considera la intervención psicosocial en las prácticas parentales de madres y padres de adolescentes como un proceso de transformación social que apunta a la promoción de la autogestión en la determinación y solución de sus necesidades, enfatizando en la necesidad de cogestionar la formación de sus hijas e hijos, sobre la base de una participación crítica que fortalece el sentido de pertenencia, identidad, así como el establecimiento de un compromiso social. Esta propuesta va sensibilizando a padres y madres con la atención a los y las adolescentes en relación con la prevención o la presencia de sintomatología depresiva, a través de la aplicación de un programa en el que se utilizan diferentes técnicas y procedimientos ya validadas por el grupo de investigación

Trypsin/α-amylase inhibitors and thionins: possible defence proteins from barley

This chapter reviews recent work on the trypsin/α-amylase inhibitor and thionin protein families. The genomic distribution of protein genes in barley and related species, gene expression and in vitro activities are considered. Some of the evidence of a possible defence role against stored products pests for inhibitors and thionins is briefly discusse