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High nitrogen use efficiency (NUE) in bread wheat is pivotal to sustain high productivity. Knowledge about the physiological and transcriptomic changes that regulate NUE, in particular how plants cope with nitrogen (N) stress during flowering and the grain filling period, is crucial in achieving high NUE. Nitrogen response is differentially manifested in different tissues and shows significant genetic variability. A comparative transcriptome study was carried out using RNA-seq analysis to investigate the effect of nitrogen levels on gene expression at 0 days post anthesis (0 DPA) and 10 DPA in second leaf and grain tissues of three Australian wheat (Triticum aestivum) varieties that were known to have varying NUEs. A total of 12,344 differentially expressed genes (DEGs) were identified under nitrogen stress where down-regulated DEGs were predominantly associated with carbohydrate metabolic process, photosynthesis, light-harvesting, and defense response, whereas the up-regulated DEGs were associated with nucleotide metabolism, proteolysis, and transmembrane transport under nitrogen stress. Protein–protein interaction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis further revealed that highly interacted down-regulated DEGs were involved in light-harvesting and photosynthesis, and up-regulated DEGs were mostly involved in steroid biosynthesis under N stress. The common down-regulated genes across the cultivars included photosystem II 10 kDa polypeptide family proteins, plant protein 1589 of uncharacterized protein function, etc., whereas common up-regulated genes included glutamate carboxypeptidase 2, placenta-specific8 (PLAC8) family protein, and a sulfate transporter. On the other hand, high NUE cultivar Mace responded to nitrogen stress by down-regulation of a stress-related gene annotated as beta-1,3-endoglucanase and pathogenesis-related protein (PR-4, PR-1) and up-regulation of MYB/SANT domain-containing RADIALIS (RAD)-like transcription fac

TaNAC-S transcription factor (TF) was previously found to have effects on senescence and grain protein content (GPC) in wheat. However, to date, its genetic architecture, association with other genes and the underlying regulatory mechanism is still unclear, making it a challenging task to utilize its variation in wheat breeding. In the current study, molecular characterization of TaNAC-S gene was conducted in 48 Australian wheat cultivars (Triticum aestivum) with the aim of improving GPC and yield in Australian wheat cultivars. Moreover, to understand the molecular function of TaNAC-S in nitrogen metabolism and how it regulates senescence, gene expression analysis was carried out using both RNA-seq and qRT-PCR using Triticum aestivum cv. Mace, Spitfire and Volcani that differ in nitrogen use efficiency (NUE) and length of maturity. As results, one copy of TaNAC-S gene was identified in each of the group 7 chromosomes (A, B and D) and mapped to the long arms of chromosomes. The coding sequences of the genes on 7A, 7B, and 7D are 870 bp, 879 bp, and 876 bp in length, respectively, and encode proteins in size of 289, 292 and 291 residues, respectively. Comparing the individual sequences in the selected cultivars, two alleles each were identified for TaNAC-S-7A and TaNAC-S-7B. No allelic variation was found for TaNACS-7D. Analysis of the translated protein sequences revealed no amino acid changes in TaNAC-S-7A alleles, whereas nucleotide changes in TaNAC-S-7B resulted in amino acid changes from threonine to methionine and leucine to valine. Moreover, gene sequences analysis of 1000 bp upstream of the TaNAC-S-7A promoter regions revealed a 36 bp insertion in 142 bp upstream of the start codon, while no significant difference was detected in the promoter sequences between TaNAC-S-7B alleles. TaNAC-S-7D promoter sequences were also found to be non-polymorphic across all cultivars. Further promoter motif analysis revealed the presence of multiple circadian, ethylene, NAC-OR