Identification and characterization of nitrate-related genes in durum wheat (Triticum turgidum L. subsp. durum Desf.) to improve Nitrogen Use Efficiency

The increasing foods demand caused by the increasing world population posed new challenges to the modern agriculture. In recent years, many strategies have been adopted to cope with these needs and satisfy these demands. Among these, the improvement of agronomic techniques and plant selection and breeding are crucial for a sustainable improvement of crop production. Nitrogen (N) is an essential nutrient for plant growth and development due to its important role in several biochemical pathways, constituting 2-5 % of plant dry matter. The excessive use of N fertilizer led to high environmental pollution with impacts on both ecosystems and human health. Therefore, N inputs reduction is one of the most important challenges to develop more sustainable agricultural systems. The selection of genotypes with a high Nitrogen Use Efficiency (NUE) is crucial to achieve this goal. Indeed, NUE is a complex trait governed by both environmental and genetic factors and their interaction. To reach a deep understanding of the molecular mechanisms involved in NUE and their role in plant selection for the complex trait is a common goal for crops. Wheat is one of the most important cereal crops worldwide due to its adaptability to a wide range of environments. Durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn. syn. Triticum durum) account for almost 5% of the total world wheat production with more than half belonging to the Mediterranean area. The recent complete durum wheat genome assembly report allows for a deeper and easier genetic investigation of more complex traits and their regulation, including NUE-related genes. A better understanding of the induction and the regulation of N-related genes might assist both breeding molecular assisted programs and the selection of novel high NUE genotypes. The high genome duplication and its being allopolyploid as well as the large size of the durum wheat genome pose further challenges for the identification of putative target genes. The aim of this work was the identification of key genes involved in N-related pathways responsible for NUE differences between genotypes and the characterization of transcriptional patterns underlying nitrate uptake, remobilization, and assimilation dynamics and their regulatory network. To achieve these goals, nine genotypes, chosen for their large variability in terms of plant growth habits, grain yield potential, and year of release were adopted for NUE evaluation (Chapter 2). Here, we focused on the identification of durum wheat genotypes highly contrasting for NUE. A first trial was performed in pots and in a controlled environment, using labeled 15N fertilizer mainly to quantify Nitrogen Uptake Efficiency (NUpE). The second trial, performed in field with high and low N supply, showed high variability for the complex trait allowing us to select four genotypes (Senatore Cappelli, Orizzonte, Antalis, and Appio) characterized for contrasting NUE. These genotypes were then used for a comparative transcriptomic analysis in a time-course design under different nitrogen availability. We further focused on the characterization of the two main gene families involved in the nitrate transport, NPF (formerly, NRT1) and NRT2, in the durum wheat genome (Chapter 3). NPF and NRT2 families are of large importance for nitrate uptake from the soil, its translocation and remobilization in different plant tissues as well as for nitrate signaling. Here, for the first time, we identified two-hundreds eleven (211) Triticum durum NPF (TdNPF) and twenty (20) TdNRT2s, providing a deep annotation of their protein sequences and conserved domains. We further described their evolutionary relationships and the putative Transcription Factors (TFs) involved in their regulation, mainly MYB and MYC and ABA related TFs for TdNRT2s and TdNPFs, respectively. High salinity in soil also affects the availability of nutrients, their uptake, translocation or portioning within the plant. Thus, we also investigated the salt-stress tolerance aided by Arbuscular Mycorrhizal Fungi (AMF) in durum wheat comparing AMF-inoculated and uninoculated plants (Chapter 4). AMF hyphae have been reported to supply up to 25% plant N, enhancing N uptake through a higher membrane stability. The study of AMF-induced genes might help to detect key genes involved in stress-response and nutrient uptake. In our experiment, the expression of genes involved in trehalose metabolism, RNA processing, vesicle trafficking, cell wall organization, and signal transduction was significantly enhanced by the AMF fungi symbiosis. Furthermore, many transcription factors, including WRKY, NAC, and MYB, known for their key role in plant abiotic stress response appeared differentially expressed between treatments. We further described the co-expression relationship between these genes and how these may affect the plant stress response. Finally, the four genotypes previously selected for their contrasting NUE, were adopted for a high-depth comparative RNA sequencing to describe the expression profiles in response to both high and low N supply to detect genotype-specific expression patterns potentially involved in their different NUE (Chapter 5). We were able to precisely supply nitrate in both high and low concentrations by using a hydroponic system, focusing on both short- (8h) and long-term (96h) plant responses to N supply. To cluster Differential Expressed Genes (DEGs) and identify functional pathways involved in nitrate metabolism, a Weighted Genes Co-expression Analysis (WGCNA) were carried out able also to detect modules of co-expression among genotypes and treatments. The comparison between contrasting genotypes allowed us to select candidate genes related to high NUE and its components. The analysis was performed on both root and shoot to break down the expression patterns involved in N uptake from the soil as well as N translocation and assimilation. Forty and thirty-four thousand DEGs were detected in root and shoot, respectively, clustered in 21 and 28 co-expression modules that were functionally characterized using GO terms enrichments. Genes involved in peptide biosynthesis, transmembrane transport, translation, oxoacid and carboxylic acid metabolisms were highly induced in root in response to N treatments whereas genes involved in photosynthesis, isoprenoid and terpenoid biosynthesis, lipid, carbohydrate and oxoacid metabolism were up-regulated in shoot. Furthermore, TFs and regulatory genes (such as Protein Kinases) involved in the N response were characterized. We also detected genotype specific expression variation inside the co-expression modules and among TdNPFs and TdNRT2s. In detail, the two older varieties, Cappelli and Appio showed a higher and faster induction of many genes related to oxidative stress response, transmembrane transport and amino acid transport as well as many TdNPFs, mainly in root. This study provided new insights into the molecular mechanisms of durum wheat in response to N supply and revealed many functional pathways involved in N uptake, translocation and assimilation in durum wheat. Furthermore, the utilization of contrasting genotypes for NUE aided the identification of several candidate genes for improving the complex trait in durum wheat.