Nutritional value and response to drought stress mediated by metabolites of lettuce (Lactuca sativa L.) and wild relatives using biochemical, genetic, genomic and transcriptomic tools
Date
2025-05-19
Authors
Medina Lozano, InésJournal Title
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texto , tesis , tesis doctoralAbstract
[ES] La lechuga (Lactuca sativa L.), una de las hortalizas de hoja más populares a nivel mundial, es ampliamente reconocida como un alimento saludable. En los últimos años, su producción ha aumentado debido probablemente al creciente interés de los consumidores por el impacto de la dieta en la salud. No obstante, los programas de mejora de lechuga se han venido centrando principalmente en la resistencia a enfermedades y en el incremento del rendimiento, dejando de lado la mejora de las propiedades beneficiosas para la salud o de la calidad nutricional. La tolerancia a estreses abióticos es otro de los aspectos olvidados en los programas de mejora de lechuga, a pesar de su importancia en el escenario actual de cambio climático. Por tanto, los objetivos de esta tesis son, por un lado, el estudio de la base genética del contenido en vitamina C, principal indicador del valor nutricional, como punto de partida para un proyecto futuro de biofortificación y, por otro lado, el estudio del impacto del estrés por sequía en el contenido en vitamina C y antocianinas, dos potentes compuestos antioxidantes, en germoplasma de lechuga y especies silvestres relacionadas. Para alcanzar estos objetivos, se cuantificó en primer lugar el contenido de estos compuestos en variedades comerciales (10) y tradicionales (13) de lechuga, así como en parientes silvestres (7). El contenido medio más alto en vitamina C se encontró en las especies silvestres y el más bajo en las variedades comerciales. Por el contrario, las variedades comerciales fueron las que mostraron la mayor concentración media de antocianinas y los parientes silvestres, la menor. Las variedades tradicionales mostraron un contenido intermedio de los dos compuestos. No obstante, teniendo en cuenta los datos brutos, el contenido más alto en vitamina C se encontró en una planta de la variedad tradicional ‘Lechuga del Pirineo’. Estos resultados sentaron las bases para las dos líneas de investigación abordadas en esta tesis. Por un lado, se seleccionó la accesión ‘Lechuga del Pirineo’ con el objetivo de obtener en el futuro una variedad uniforme rica en vitamina C. La homogeneidad genética se alcanzó en la población S1 y el contenido en ácido ascórbico, la forma de vitamina C con mayor actividad biológica, aumentó significativamente en la población S2 (S1 y S2 se obtuvieron por autofecundación de la planta más rica en vitamina C de la población original S0 y de S1, respectivamente). Se llevó a cabo un estudio de asociación del genoma completo del contenido en vitamina C en 205 plantas de la población S0 de ‘Lechuga del Pirineo’ y un panel de diversidad de 21 variedades cultivadas. Se encontraron asociaciones genéticas significativas entre 17 SNPs (single nucleotide polymorphisms) y el contenido en ácido dehidroascórbico (la otra forma de vitamina C) en una región de 5,1 Mb del cromosoma 2. Entre ellos, 12 mostraron un desequilibrio de ligamiento alto con el SNP más significativamente asociado. Se identificaron genes putativamente relacionados con el contenido en vitamina C en esa región, incluyendo algunos cuya participación en la regulación de este compuesto se ha descrito previamente en otros cultivos, como tomate, patata y trigo. Por otro lado, se seleccionaron cinco variedades de lechuga cultivada y dos parientes silvestres para evaluar el efecto del estrés por sequía en el contenido de vitamina C y antocianinas durante dos años. El déficit hídrico provocó un aumento en el contenido en antocianinas en todas las accesiones estudiadas, a diferencia de lo ocurrido con la vitamina C, que disminuyó en todos los casos. Esto sugiere que las antocianinas podrían estar implicadas en la tolerancia a sequía en algunas especies de Lactuca y, por este motivo, los análisis transcriptómicos se centraron en la búsqueda de genes diferencialmente expresados (DEGs) relacionados específicamente con las antocianinas bajo condiciones de estrés hídrico. Una variedad comercial de lechuga (‘Romired’) y una especie silvestre (Lactuca homblei De Wild) fueron seleccionadas para el estudio transcriptómico (RNA-seq). Entre las accesiones estudiadas, L. homblei fue la que mostró el mayor enriquecimiento en el contenido de antocianinas (y la única en la que fue significativo estadísticamente), así como la mayor respuesta a nivel transcriptómico bajo condiciones de estrés. El número de DEGs activados, incluyendo aquellos relacionados con el contenido en antocianinas, fue mayor en L. homblei, mientras que el número de DEGs reprimidos fue mayor en ‘Romired’. Entre los DEGs que mostraron el mayor cambio en los niveles de expresión y una correlación significativa tanto con el contenido en antocianinas como con el tratamiento de estrés por sequía, se seleccionaron 19 genes candidatos. Cabe destacar que estos genes mostraron expresión diferencial únicamente en L. homblei. Además, todos ellos han mostrado funciones relacionadas con el contenido en antocianinas y/o la respuesta a estreses bióticos y abióticos en otras plantas. En las secuencias de la especie silvestre, algunos genes candidatos presentaron polimorfismos con impactos predichos altos o moderados en la función de sus proteínas, como una proteína de choque térmico que participa en el cierre estomático inducido por ácido abscísico, un factor de transcripción involucrado en la respuesta a estreses bióticos y abióticos y una fosfolipasa implicada en la acumulación de antocianinas bajo estrés abiótico. Para validar su expresión mediante PCR cuantitativa (qPCR), se seleccionó previamente el gen de referencia más estable para el experimento de estrés por sequía en base a los datos de RNA-seq. Los patrones de expresión obtenidos con las dos técnicas (RNA-seq y qPCR) fueron consistentes y, por tanto, se pudo confirmar la fiabilidad del estudio transcriptómico. Considerando todos los resultados, L. homblei podría ser una fuente valiosa de tolerancia a estrés por sequía y de compuestos beneficiosos para la salud, lográndose también un cultivo de lechuga más sostenible.
[EN] Lettuce (Lactuca sativa L.), one of the most popular leafy vegetables worldwide, is widely considered a healthy food. In recent years, its production has increased likely due to the growing interest of consumers in the impact of diet on health. However, lettuce breeding programmes have mainly been focused on disease resistance and yield increase, putting aside the enhancement of health beneficial properties or nutritional quality. Tolerance to abiotic stresses is another neglected aspect in lettuce breeding programmes, despite its importance in the current climate change scenario. Therefore, the objectives of this thesis are, on one hand, to study the genetic basis of vitamin C content, the main indicator of nutritional value, as the beginning of a future biofortification project and, on the other hand, to study the drought stress impact on vitamin C and anthocyanin content, two powerful antioxidant compounds, in lettuce germplasm and wild relative species. To achieve these goals, the content of these compounds was firstly quantified in commercial (10) and traditional (13) varieties, as well as in wild relatives (7). The highest average content in vitamin C was found in the wild species and the lowest in the commercial varieties. On the contrary, the commercial varieties showed the highest average concentration of anthocyanins and the wild relatives, the lowest. The traditional varieties showed an intermediate content of both compounds. Nevertheless, considering raw data, the highest content in vitamin C was found in one plant of the traditional variety ‘Lechuga del Pirineo’. These results served as the starting point for the two research lines pursued in the current thesis. On one hand, the accession ‘Lechuga del Pirineo’ was selected with the aim of eventually obtaining a uniform variety rich in vitamin C. Genetic homogeneity was already reached in S1 population and ascorbic acid, the vitamin C form with the highest biological activity, increased significantly in S2 population (S1 and S2 were obtained by self-pollinating the plant richest in vitamin C from the original S0 and S1 populations, respectively). A genome-wide association study of vitamin C content was conducted in 205 plants of ‘Lechuga del Pirineo’ S0 and a diversity panel of 21 cultivated varieties. Significant genetic associations were found between 17 single nucleotide polymorphisms (SNPs) and the content in dehydroascorbic acid (the other form of vitamin C) in a 5.1 Mb region of chromosome 2. Among them, 12 showed high linkage disequilibrium with the most significantly associated SNP. Genes putatively related to vitamin C content were identified in that region, including some that have previously been described to participate in the regulation of this compound in other crops, such as tomato, potato and wheat. On the other hand, a set of five cultivated lettuce varieties and two wild relatives were selected to evaluate the effect of drought stress on both vitamin C and anthocyanin content for two years. Water deficit caused an increase in anthocyanin content in all the assayed accessions, unlike what happened with vitamin C amount, which decreased in all cases. This suggests that anthocyanins might be implied in drought tolerance in some Lactuca spp. and, for this reason, transcriptomic analyses were focused on the identification of differentially expressed genes (DEGs) specifically related to anthocyanins under water stress conditions. A commercial lettuce variety (‘Romired’) and a wild species (Lactuca homblei De Wild) were selected for the transcriptomic study (RNA-seq). L. homblei was the accession that showed both the highest enrichment in anthocyanin content (and the only in which it was statistically significant) among all the tested ones and the highest transcriptomic response when subject to stress. The number of upregulated DEGs, including those related to anthocyanin content, was higher in L. homblei, whereas the number of downregulated DEGs was higher in ‘Romired’. Among DEGs showing the highest change in the expression level and a significant correlation with both anthocyanin content and drought stress treatment, 19 candidate genes were selected. Interestingly, these genes resulted to be differentially expressed exclusively in L. homblei. In addition, all of them were shown to have functions related to anthocyanin content and/or response to biotic and abiotic stresses in other plants. In the wild species sequences, some candidate genes had polymorphisms with high or moderate predicted impacts on their protein functions, like a heat shock protein that participates in the abscisic acid-induced stomatal closure, a transcription factor involved in responses to biotic and abiotic stresses and a phospholipase implied in anthocyanin accumulation under abiotic stress. To validate their expression by quantitative PCR (qPCR), the most stable reference gene for the drought stress experiment was previously selected based on the RNA-seq data. Expression patterns from both techniques (RNA-seq and qPCR) were consistent and, thus, the reliability of the transcriptomic study was confirmed. All results considered, L. homblei could be a valuable resource in lettuce breeding programmes as a source of drought stress tolerance and health beneficial compounds, making also possible a more sustainable lettuce cultivation.
[EN] Lettuce (Lactuca sativa L.), one of the most popular leafy vegetables worldwide, is widely considered a healthy food. In recent years, its production has increased likely due to the growing interest of consumers in the impact of diet on health. However, lettuce breeding programmes have mainly been focused on disease resistance and yield increase, putting aside the enhancement of health beneficial properties or nutritional quality. Tolerance to abiotic stresses is another neglected aspect in lettuce breeding programmes, despite its importance in the current climate change scenario. Therefore, the objectives of this thesis are, on one hand, to study the genetic basis of vitamin C content, the main indicator of nutritional value, as the beginning of a future biofortification project and, on the other hand, to study the drought stress impact on vitamin C and anthocyanin content, two powerful antioxidant compounds, in lettuce germplasm and wild relative species. To achieve these goals, the content of these compounds was firstly quantified in commercial (10) and traditional (13) varieties, as well as in wild relatives (7). The highest average content in vitamin C was found in the wild species and the lowest in the commercial varieties. On the contrary, the commercial varieties showed the highest average concentration of anthocyanins and the wild relatives, the lowest. The traditional varieties showed an intermediate content of both compounds. Nevertheless, considering raw data, the highest content in vitamin C was found in one plant of the traditional variety ‘Lechuga del Pirineo’. These results served as the starting point for the two research lines pursued in the current thesis. On one hand, the accession ‘Lechuga del Pirineo’ was selected with the aim of eventually obtaining a uniform variety rich in vitamin C. Genetic homogeneity was already reached in S1 population and ascorbic acid, the vitamin C form with the highest biological activity, increased significantly in S2 population (S1 and S2 were obtained by self-pollinating the plant richest in vitamin C from the original S0 and S1 populations, respectively). A genome-wide association study of vitamin C content was conducted in 205 plants of ‘Lechuga del Pirineo’ S0 and a diversity panel of 21 cultivated varieties. Significant genetic associations were found between 17 single nucleotide polymorphisms (SNPs) and the content in dehydroascorbic acid (the other form of vitamin C) in a 5.1 Mb region of chromosome 2. Among them, 12 showed high linkage disequilibrium with the most significantly associated SNP. Genes putatively related to vitamin C content were identified in that region, including some that have previously been described to participate in the regulation of this compound in other crops, such as tomato, potato and wheat. On the other hand, a set of five cultivated lettuce varieties and two wild relatives were selected to evaluate the effect of drought stress on both vitamin C and anthocyanin content for two years. Water deficit caused an increase in anthocyanin content in all the assayed accessions, unlike what happened with vitamin C amount, which decreased in all cases. This suggests that anthocyanins might be implied in drought tolerance in some Lactuca spp. and, for this reason, transcriptomic analyses were focused on the identification of differentially expressed genes (DEGs) specifically related to anthocyanins under water stress conditions. A commercial lettuce variety (‘Romired’) and a wild species (Lactuca homblei De Wild) were selected for the transcriptomic study (RNA-seq). L. homblei was the accession that showed both the highest enrichment in anthocyanin content (and the only in which it was statistically significant) among all the tested ones and the highest transcriptomic response when subject to stress. The number of upregulated DEGs, including those related to anthocyanin content, was higher in L. homblei, whereas the number of downregulated DEGs was higher in ‘Romired’. Among DEGs showing the highest change in the expression level and a significant correlation with both anthocyanin content and drought stress treatment, 19 candidate genes were selected. Interestingly, these genes resulted to be differentially expressed exclusively in L. homblei. In addition, all of them were shown to have functions related to anthocyanin content and/or response to biotic and abiotic stresses in other plants. In the wild species sequences, some candidate genes had polymorphisms with high or moderate predicted impacts on their protein functions, like a heat shock protein that participates in the abscisic acid-induced stomatal closure, a transcription factor involved in responses to biotic and abiotic stresses and a phospholipase implied in anthocyanin accumulation under abiotic stress. To validate their expression by quantitative PCR (qPCR), the most stable reference gene for the drought stress experiment was previously selected based on the RNA-seq data. Expression patterns from both techniques (RNA-seq and qPCR) were consistent and, thus, the reliability of the transcriptomic study was confirmed. All results considered, L. homblei could be a valuable resource in lettuce breeding programmes as a source of drought stress tolerance and health beneficial compounds, making also possible a more sustainable lettuce cultivation.
Description
Esta tesis doctoral se presenta como un compendio de cinco artículos de investigación previamente publicados. Las publicaciones que constituyen el cuerpo principal de la tesis se detallan a continuación:
- Medina-Lozano, I, Bertolín, JR, Díaz, A (2021). Valor nutricional de la lechuga comercial y tradicional (Lactuca sativa L.) y parientes silvestres: contenido de vitamina C y antocianinas. Food Chem. 359, 129864. https://doi.org/10.1016/j.foodchem.2021.129864
- Medina-Lozano, I, Bertolín, JR, Plieske, J, Ganal, M, Gnad, H, Díaz, A (2024b). Estudios de diversidad genética y asociación del genoma completo para el contenido de vitamina C en lechuga (Lactuca sativa L.) utilizando matrices SNP de alto rendimiento. Plant Genome 17, e20518. https://doi.org/10.1002/tpg2.20518
- Medina-Lozano, I, Bertolín, JR, Díaz, A (2024a). Impacto del estrés por sequía en el contenido de vitamina C y antocianinas en lechugas cultivadas (Lactuca sativa L.) y parientes silvestres (Lactuca spp.). Front. Plant Sci. 15, 3389. https://doi.org/10.3389/fpls.2024.1369658
- Medina-Lozano, I, Arnedo, MS, Grimplet, J, Díaz, A (2023). Selección de nuevos genes de referencia mediante RNA-Seq y su evaluación para normalizar datos de expresión de qPCR en genes relacionados con antocianinas en lechuga y parientes silvestres. Int. J. Mol. Sci. 24, 3052. https://doi.org/10.3390/ijms24033052
- Medina-Lozano, I, Grimplet, J, Díaz, A (2025). Aprovechando la diversidad de un pariente silvestre de la lechuga para identificar genes relacionados con antocianinas que responden transcripcionalmente al estrés por sequía. Front. Plant Sci. 15, 1494339. https://doi.org/10.3389/fpls.2024.1494339
Keywords
Bibliographic citation
Medina-Lozano, I. (2025). Nutritional value and response to drought stress mediated by metabolites of lettuce (Lactuca sativa L.) and wild relatives using biochemical, genetic, genomic and transcriptomic tools. [Tesis doctotal, Universidad de Zaragoza]. https://hdl.handle.net/10532/
AGROVOC subjects
LechugaVitamin c
Antocianina
Estrés de sequia
Fortificación de alimentos
Sponsorship
Este trabajo fue financiado por los proyectos RTA2017-00093-00-00 del Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria; LMP164_18 y LMP148_21 del Gobierno de Aragón; PID2022-138484OR-I00 del Ministerio de Ciencia e Innovación (MCIN) y la Agencia Estatal de Investigación (AEI); AGROALNEXT del MCIN con financiación de la Unión Europea NextGenerationEU: PRTR-C17.11; y por el Programa Operativo FEDER Aragón 2014-2020, 2020-2022 y 2023-2025, y el Fondo Social Europeo de la Unión Europea (A12-17R, A12_20R y A12_23R: “Grupo de investigación en fruticultura: caracterización, adaptación y mejora genética”). Inés Medina Lozano fue apoyada por el contrato predoctoral para la formación de doctores PRE2018-084987 del Ministerio de Ciencia, Innovación y Universidades y la Agencia Estatal de Investigación.