Expanding the TaNAS gene family in bread wheat and exploring potential 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) for long-distance transport. We identified 34 TaNAS genes in bread wheat ( Triticum aestivum L., cv. Chinese Spring), and four additional cultivar-specific TaNAS genes, representing the largest NAS gene family identified to date. The expression of all TaNAS genes was highest in roots and upregulated (apart from the TaNAS9 homoeologs) in response to hydroponic Fe deficiency. The TaNAS proteins ranged between 180 and 384 amino acids in length and showed variable N- and C-termini. To understand TaNAS function, we transformed bread wheat cv. Fielder to overexpress coding sequences from either TaNAS1 , TaNAS3 , TaNAS4 , TaNAS6 , or TaNAS7 , and isolated three single locus homozygous events and null segregant (NS) controls for each TaNAS overexpression line 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 without any negative impacts on agronomic traits. These results demonstrate the usefulness of available online genomic resources for analysing the TaNAS gene family in bread wheat and highlight intragenic strategies to enhance grain nutritional quality. Significance statement Nicotianamine synthase ( NAS ) genes play key roles in metal transport and homeostasis in plants. This study describes the identification of 34 TaNAS genes in bread wheat, representing a significant expansion on previous TaNAS studies, and demonstrates that constitutive overexpression of specific TaNAS genes could provide a novel intragenic approach for iron and zinc biofortification of bread wheat.