Caracterización fisiológica, bioquímica y molecular del metabolismo del carbono y nitrógeno en plantas de trigo duro (Triticum turgidum ssp. durum) crecidas en CO2 elevado con distinta disponibilidad de nitrógeno

Abstract

[EN] Climate change is a major challenge to food security, thus it is important to understand how future crops can be bred to withstand adverse environmental conditions. Growth in elevated CO2 often leads to a down-regulation of photosynthesis and a loss of nitrogen in plant tissues, indicative of a shift in the carbon-nitrogen balance of the plant and highlighting the importance of studying the tight interaction between both metabolic pathways. The overall aim of this Doctoral Thesis is to understand the mechanisms involved in the decline of N found in plants grown in elevated CO2 through the integrated investigation of biomass, gene expression, possible post-translational modifications, enzyme activities and levels of metabolites and other compounds of the plants. To achieve this goal (a) a qRT-PCR platform for analysing the expression of carbon and N metabolism genes in durum wheat has been developed; (b) it has been analysed whether growth in elevated CO2 limits N uptake when nutrient movement to roots is unrestricted, by using hydroponic cultures; (c) whether N availability and plant development modify the effect of CO2 enrichment on N uptake and allocation within plants has been assessed; and (d) the transcriptional response to elevated [CO2] and high temperature at two N supplies in durum wheat grown in field chambers, and its relationship with other biochemical and physiological parameters has also been investigated. In this research, the qRT-PCR primer platform developed has enabled us to measure transcript levels in leaves for 125 genes and in roots for 38 genes. In wheat grown in hydroponic culture, elevated CO2 did not affect the nitrate uptake per unit root mass at anthesis, irrespective of N availability in the nutrient solution, but decreased it at early grain filling in plants with suboptimal N supply. Photosynthetic acclimation to elevated CO2 was found in plants with superoptimal, but not with suboptimal N supply, in association in the latter with an improvement of leaf N status induced by elevated CO2, which was accompanied by the induction of genes for photosynthesis and N assimilation, and increases of Rubisco protein and activity, and amino acid and protein contents. In contrast, in plants with superoptimal N supply, the decline of photosynthetic capacity was related to a loss of Rubisco protein and a limitation in photosynthetic electron transport due to the inhibition of N assimilation. However, when N availability was more restricted, growth at elevated CO2 led to a down-regulation of photosynthesis in N deficient plants but not in those with optimal N supply, probably because with the latter N assimilation was not inhibited, organic nitrogen content was not reduced in flag leaves, and a strong up-regulation of nitrogen metabolism genes occurred in the roots. In N deficient plants, elevated CO2 increased the biomass of the plants and the carbohydrate content in the flag leaf, but sharply decreased the foliar levels of ATP, ADP, NADP, RuBP, FBP, glycolytic and tricarboxylic acid cycle intermediates and amino acids, and also repressed genes for N assimilation and induced a decline of N concentration per unit flag leaf area. When durum wheat was grown in field chambers, elevated CO2 also caused down-regulation of photosynthetic capacity and loss of N compounds, including Rubisco, associated with a repression of genes involved in photosynthesis and N assimilation, particularly at low N supply. High temperatures increased stomatal conductance and thus did not inhibit photosynthesis, even though Rubisco protein and activity, soluble protein and leaf N were decreased and gene expression for photosynthesis and N assimilation was also repressed. The results from the hydroponic experiments suggest that elevated CO2 restricts N uptake late in development, superoptimal N supply overriding this restriction. Increased N allocation to the shoot, mainly to the flag leaf, at suboptimal N supply could alleviate photosynthetic acclimation to elevated CO2. When N availability was more restricted by reducing frequency of renewal as well as concentration of the nutrient solution, the photosynthesis in N deficient plants was limited by ribulose-1,5-bisphosphate carboxylation and regeneration. Finally, the results of the field experiment showed that under future Climate Change scenario, C fixation capacity and N assimilation will be down-regulated in an N supply-dependent extent. This investigation can contribute to higher and more stable crop yield of durum wheat in the face of Climate Change, and can suggest criteria that potentially can be used directly in conventional breeding programmes.

[ES] El cambio climático es un desafío importante para la seguridad alimentaria, por lo que es importante entender cómo los cultivos futuro pueden ser criados para soportar condiciones ambientales adversas. El crecimiento en niveles elevados de CO2 a menudo conduce a una baja regulación de la fotosíntesis y la pérdida de nitrógeno en los tejidos vegetales, indicativo de un cambio en el balance carbono-nitrógeno de la planta, lo que pone de relieve la importancia de estudiar la interacción estrecha entre las dos vías metabólicas. El objetivo general de esta Tesis Doctoral es comprender los mecanismos implicados en la disminución de N encontrado en las plantas cultivadas en niveles elevados de CO2 a través de la investigación integrada de biomasa, la expresión génica, las posibles modificaciones posteriores a la traducción, las actividades enzimáticas y los niveles de metabolitos y otros compuestos de las plantas. Para lograr este objetivo (a) se ha desarrollado una plataforma de QRT-PCR para el análisis de la expresión de genes del metabolismo de carbono y N en trigo duro; (B) se ha analizado si el crecimiento en los límites elevados de CO2 N captación cuando el movimiento de nutrientes a las raíces no está restringido, mediante el uso de cultivos hidropónicos; (C) si la disponibilidad de N y desarrollo de las plantas modifican el efecto del enriquecimiento de CO2 en la absorción de N y la asignación dentro de las plantas se ha evaluado; y (d) la respuesta transcripcional a elevada [CO2] y la alta temperatura a dos fuentes de N en trigo duro cultivado en cámaras de campo, y su relación con otros parámetros bioquímicos y fisiológicos también se ha investigado. Esta investigación puede contribuir a un mayor y más estable rendimiento de los cultivos de trigo duro de cara al cambio climático, y pueden sugerir criterios que potencialmente pueden ser utilizados directamente en los programas convencionales de mejoramiento.