Analysis of the <i>TaNAS</i> gene family in bread wheat identifies additional members and candidates for intragenic biofortification

Higher plants utilise nicotianamine synthase (NAS) enzymes to produce nicotianamine (NA), a non-protein amino acid that chelates metals such as iron (Fe) and zinc (Zn) and plays key roles in metal transport and homeostasis. In this study we identified 34 TaNAS genes in the recently annotated cv. Chinese Spring bread wheat genome (IWGSC Refseq 1.1 and 2.1), as well as four cultivar-specific TaNAS genes in the 10+ Wheat Genome Project, representing a significant expansion on previous analyses of the TaNAS gene family. The majority of TaNAS genes were highly expressed in roots and upregulated in response to hydroponic Fe deficiency. The TaNAS proteins ranged from 180 to 384 amino acids in length and showed variable N- and C-termini. To investigate TaNAS function we transformed bread wheat cv. Fielder with constitutive overexpression constructs of TaNAS1, TaNAS3, TaNAS4, TaNAS6, or TaNAS7, and isolated single locus homozygous events and null segregant (NS) controls for glasshouse and field evaluation. Under field conditions, grain Fe, Zn and NA concentrations were up to 1.6-fold, 1.8-fold and 3.7-fold higher, respectively, in TaNAS6 events relative to NS with no detrimental impact on agronomic traits. Transient expression analyses in Nicotiana benthamiana demonstrated that the TaNAS6 enzyme produced significantly more NA in leaf tissue relative to other NAS enzymes. These results broaden our understanding of the TaNAS gene family in bread wheat and highlight novel biofortification strategies to improve grain nutrition.