TmNAS3 from Triticum monococum directly regulated by TmbHLH47 increases Fe content of wheat grain

Biofortification is an effective way to enhance wheat grain Fe content. However, Fe overload inhibits the growth and development of wheat. In this work, the impact of Triticum monococcum nicotianamine synthase 3 (TmNAS3) on Fe accumulation in wheat grain was analyzed. Transgenic wheat expressing TmNAS3 was obtained via Agrobacterium-mediated transformation. The concentrations of Fe in the grains of two transgenic wheat lines were 62.42 μg/g and 68.75 μg/g, while that in the non-transgenic line (NT) was only 29.51 μg/g. Exogenous Fe application induced the expression of natural resistance-associated macrophage protein 3 (NRAMP3), NRAMP6, yellow stripe-like protein 3 (YSL3), YSL6, and vacuolar iron transporter 2 in transgenic wheat. The transcription factor that bound to the TmNAS3 promoter was identified, and the findings suggested that TmbHLH47 directly interacted and promoted the transcription of TmNAS3. Moreover, TmbHLH47 was observed to bind directly to the G-box in TmNAS3 promoter and regulated the transcriptional level of TmNAS3. Our findings contribute a TmbHLH47/TmNAS3 transcriptional pathway and thereby provide a potential strategy for improving the Fe concentration of wheat through genetic engineering.