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The phenolic components of virgin olive oil (VOO) are key to the organoleptic and functional quality of this emblematic food of the Mediterranean diet. Olive breeding for improving VOO phenolic fraction, including both phenolic compounds with secoiridoid structures and tocopherols, depends largely on the identification and characterization of the key genes determining the biosynthesis of these compounds in the olive fruit as well as those that transform these phenolic components during the oil extraction process. However, there is very little knowledge about the key genes/enzymes controlling these metabolic processes. In this work, the role of olive polyphenol oxidase (PPO) in the metabolism of olive phenolic compounds has been studied. Three PPO genes have been identified from a transcriptome obtained from six olive cultivars with highly contrasting phenolic contents. These genes were expressed in Escherichia coli and the functional identity of the recombinant proteins verified using olive phenolic substrates. The OePPO1 and OePPO2 proteins exhibit diphenolase activity, while OePPO3 is a tyrosinase that not only possesses diphenolase but also monophenolase activity, able to catalyse the hydroxylation of tyrosol to form hydroxytyrosol. The role of these PPO genes in the metabolism of phenolic compounds in the olive is analysed and the potential use of this information for olive breeding is discussed.

Virgin olive oil (VOO) represents the main source of lipids in the Mediterranean diet, its phenolic fraction being one of the main contributors to the health-promoting properties of this diet. Phenolic composition of VOO is directly related to the content of phenolic glycosides present in the olive fruit, which undergo a series of modifications during the oil extraction process. The vast majority of these phenolic glycosides comprise phenylethanoid derivatives of tyrosol and hydroxytyrosol whose metabolic origin are the hydroxylated aromatic amino acids 4-hydroxyphenylalanine (tyrosine) and 3,4-dihydroxyphenylalanine (L-DOPA). However, the synthesis of phenolic compounds in plants has traditionally been linked to the action of the phenylalanine ammonia-lyase (PAL) enzymes on the non-hydroxylated aromatic amino acid phenylalanine, thus constituting the first step in the biosynthetic pathway of different families of phenolic compounds known as phenylpropanoids. In this research, the implication of PAL in the conformation of the phenolic profile of olive fruit and VOO has been studied through the combined analysis of phenolic composition, PAL activity and gene expression in the mesocarp of the fruit from olive cultivars whose oils have very contrasting phenolic contents. According to the data, the role of PAL would be restricted to the most advanced stages of the fruit ripening process, controlling the channeling of phenylalanine towards the synthesis of phenylpropanoids, mainly anthocyanins, and thus reducing the metabolisation of tyrosine and L-DOPA towards the synthesis of phenylethanoids. Consequently, it is advisable not to use the selection of olive cultivars based on molecular markers related to PAL genes in olive breeding programs aimed at obtaining cultivars that give rise to oils with improved functional properties.

4 Páginas, Los componentes fenólicos más importantes del aceite de oliva virgen (AOV) son los derivados secoiridoideos, en cuya estructura se combinan un resto terpénico y uno fenólico. El contenido de estos compuestos en el AOV está condicionado por el contenido de glucósidos fenólicos secoiridoideos presentes en la aceituna, que son posteriormente modificados durante el proceso de extracción del aceite. Los principales glucósidos fenólicos de la aceituna, oleuropeína, ligustrósido y demetiloleuropeína, se conocen genéricamente como feniletanoides debido a la presencia de tirosol (Ty) o hidroxitirosol (Hty) en sus estructuras químicas. La biosíntesis de estos dos alcoholes fenólicos parece ser el factor limitante para la formación y acumulación de glucósidos secoiridoideos en la aceituna. En este trabajo se discute la relevancia de algunos genes/enzimas identificados y caracterizados en nuestro grupo en relación con el metabolismo del Ty e Hty como la aldehído aromático sintasa (AAS), fenilacetaldehido reductasa (PAR) o una tirosinasa (TYR) recientemente caracterizada, y se discute su posible utilidad como marcadores moleculares para la selección de variedades de olivo con contenidos fenólicos mejorados en sustitución de otros utilizados tradicionalmente como la fenilalanina amonio liasa (PAL)., La investigación reflejada en este trabajo ha sido financiada por los proyectos AGL2015-67652 y PID2020-115853RR-C32 (MCIN/AEI/10.13039/501100011033).

1 Página, El aceite de oliva virgen extra (AOVE) cuenta en su composición con un gran número de compuestos minoritarios relacionados con sus propiedades saludables, entre ellos, los compuestos fenólicos. Dichos compuestos están asimismo relacionados con la estabilidad y propiedades organolépticas del AOVE, siendo, por tanto, constituyentes de primordial importancia con respecto a la calidad comercial. La composición fenólica del AOVE depende en gran medida de la composición inicial en dichos componentes de los frutos, que posteriormente sufre una serie de transformaciones enzimáticas durante el proceso de extracción en la almazara. En el presente trabajo se compara la variabilidad del contenido y composición en fenoles en frutos recolectados en dos épocas en seis variedades (‘Arbequina’, ‘Coratina’, ‘Hojiblanca’, ‘Koroneiki’, ‘Martina’ y ‘Picual’) cultivadas en condiciones contrastantes con clima mediterráneo de Córdoba y subtropical de Tenerife, con el objetivo de estudiar la influencia de los factores genético y ambiental en la composición fenólica de los frutos. En todos los casos se ha observado un descenso acusado del contenido total de fenoles en la fecha de recolección más tardía (Figura 1), descenso que afectó de manera similar a todos los grupos de compuestos analizados que no variaron significativamente su proporción en las dos fechas analizadas (Tabla 1). El contenido total de fenoles estuvo significativamente influenciado por ambos factores: variedad, con los valores más altos en ‘Coratina’ y localidad, con valores más altos en Tenerife, sin que exista interacción entre ambos factores (Tabla 2, Figura 1). En cuanto a la composición fenólica, en general se observó un efecto genético (variedad) más importante que el ambiental para todos los grupos de compuestos (Tabla 2). En particular, aunque no hubo diferencias significativas entre variedades para los tres grupos principales de compuestos fenólicos (derivados de hidroxitirosol, derivados de tirosol y flavonas), sí que las diferencias fueron muy importantes considerando el porcentaje de compuestos individuales dentro de cada grupo principal (Figura 2). En resumen, los resultados obtenidos muestran un efecto mayoritario del genotipo (variedad) en la expresión de la composición fenólica en fruto, más importante que la variabilidad asociada al ambiente y la interacción genotipo x ambiente. Ello puede permitir simplificar la selección de genotipos de interés y señala las posibilidades de la mejora genética para obtener nuevas variedades con un contenido y composición fenólica óptimo., Ayuda PID2020-115853RR-C3 financiada por MCIN/AEI/10.13039/501100011033 y “FEDER Una manera de hacer Europa”

Resumen del trabajo presentado en la 6th International Conference on the Olive Tree and Olive Products, celebrada en Sevilla (España) del 15 al 19 de octubre de 2018., The wide olive genetic patrimony has revealed high variability for most of the agronomic and oil quality traits of interest in olive growing. Few studies, however, have addressed the interaction of this variability with the environment, a subject of particular interest considering the natural high instability of the Mediterranean climate and the challenge of the predicted climate change. The current work presents results on the interaction between genotype and environment from multi-environment trials of olive cultivars and breeding selections, planted in different edaphoclimatic conditions of Andalusia, Southern Peninsular Spain and Canary Islands. For most of the agronomic and oil quality characters evaluated (flowering phenology, flower quality, pattern of oil accumulation, fatty acid composition and phenol content and composition), significant genotype and environment effects have been observed. For example, olive cultivars grown in Tenerife under much milder winter temperatures than in the Iberian Peninsula showed substantially earlierflowering and oil accumulation. Only in the case of flowering phenology was no significant genotype effect found. Furthermore, a strong genotype x environment effect was highly consistent in all characters considered. Regarding resistance to disease, such as Verticillium wilt, the variability of results from both natural and artificial inoculations also tends to indicate a considerable environmental effect and the need for careful testing of disease evolution. All this information strongly suggests the necessity of comparative trials of olive cultivars for both adequate choice of cultivar and final selection in breeding programs.

6 Figuras.-- 1 Tabla, Different scientific evidences suggest that the long term dietary consumption of virgin olive oil (VOO) seems to be related to an attenuation of the inflammatory response and reduction of the associated risk of chronic inflammatory disease states. VOO phenolic compounds are claimed to be the main responsible for these positive health benefits. They are mainly synthetized from phenolic glucosides present in the olive fruit by the action of glucosidases occurring when they come together once the olive fruit is crushed during olive oil extraction. The genetic variability of the major phenolic compounds was studied in a representative sample of olive cultivars (Olea europaea L.) from the World Olive Germplasm Collection established at IFAPA Centre “ Alameda del Obispo in Cordoba, Spain. The most abundant phenolic components found in VOO are the secoiridoid derivatives resulting from the enzymatic hydrolysis of oleuropein, ligstroside and demethyloleuropein present in the fruit, which showed to be on average their main phenolic glucosides. The mean content of phenolic compounds in the oils was 494.51 µg/g oil, displaying a variability range of 63.74-1432.04 µg/g oil. The mean content of phenolic compounds in the fruits was 12384.23 µg/g fruit with a range of 3754.13-30696.39 µg/g fruit. Total phenolic compounds in the fruits and the oils were significantly correlated (r = 0.66). Thus, the composition and biochemical status of the olive fruit seems to be the most important variables determining the synthesis of the VOO phenolic compounds during the oil extraction process. On the other hand, the content of oleuropein and derivatives in the oils and fruits showed a correlation coefficient (r = 0.64) lower than that observed for ligstroside and derivatives (r = 0.73). These findings might be related to the higher oxidation rates of the former due to the action of oxidative enzymes during the oil extraction process as a consequence of the orthodiphenolic structure they possess. Data on phenolic composition would be of interest for the selection of optimal parents in olive breeding programs with the aim of obtaining new cultivars with improved nutritional quality

Virgin olive oil (VOO) represents the primary dietary lipid source in the Mediterranean diet and has been linked to its positive health benefits. VOO metabolites responsible for its nutritional and organoleptic quality include certain components that are transferred almost unchanged from fruit to oil (fatty acids and tocopherols) and other compounds that are synthesized during the oil extraction process by endogenous enzymes from precursors already present in the fruit (volatile and phenolic compounds). The health - promoting characteristics of VOO seems to be related to the high level of oleic acid, an adequate balance of saturated/unsaturated fatty acids, the presence of tocopherols and, specially, the contents of phenolic compounds. Meanwhile, volatile compounds are responsible for its aroma, whereas phenolic compounds are also responsible for the bitter and pungent taste notes of this oil. The final content of these metabolites related to VOO quality is controlled by the levels of expression/activity of the corresponding genes/enzymes involved in their biosynthesis which are cultivar dependent. Given that oil quality is one of the main objectives of the breeding programs aiming at obtaining new olive cultivars, knowledge of the content of these key components for the VOO quality might be very useful for the selection. Moreover, the combination of biochemical and molecular studies would allow to identify the most important genes/enzymes in relation to the oil quality and to know their regulatory mechanisms, so that this knowledge may be used in the molecular marker assisted selection of olive clones with improved oil quality features. For this purpose, the natural variability of the olive species was assessed in a collection of cultivars from the World Olive Germplasm Bank (WOGB) located at Córdoba, and the progeny of a cross of Picual x Arbequina olive cultivars. Analytical data showed that the cultivar collection displayed, in general, a higher level of variability than the progeny cross for most of the metabolites assessed, and that a single cross of olive cultivars might apparently provide enough degree of variability for most of the oil quality traits sought in a breeding program.

El estudio de la composición aromática de Fresas de la variedad Chandler, mediante GC-MS y HPLC-MS, identifica dos grupos de compuestos fundamentales en el aroma de esta variedad: Los derivados del Furaneol y los Esteres de Etilo. La formación de esteres volátiles es ... tá determinada por la disponibilidad de precursores y por las características de la enzima Alcohol Aciltransferasa. El análisis de aminoácidos libres durante la maduración del fruto muestra una relación directa entre el contenido de Alanina libre y la producción de Esteres de Etilo. La formación de esteres esta catalizada por la enzima Alcohol Aciltransferasa que se purifico y caracterizo. La enzima tiene un peso molecular de 70KDA, Phoptimo de 8 y temperatura optima de 35º C. Los estudios de especificidad indican que el Hexanol y el Acetil-coa son los substratos preferidos, los datos de especificidad por distintos alcoholes y Acil-coas indican un papel clave de la enzima en el control de la biogénesis del aroma. El primer objetivo de esta Tesis es el análisis y la caracterización de los compuestos volátiles responsables del aroma de las fresas de la variedad Chandler. En una segunda fase del estudio, se procederá a estudiar la evolución de estos compuestos y sus posibles precursores durante la maduración del fruto. La última fase del trabajo, abordará el estudio más específico de los sistemas enzimáticos implicados en la biogénesis de los principales compuestos volátiles identificados.