A two-gene strategy increases iron and zinc concentrations in wheat flour, improving mineral bioaccessibility

Abstract

Dietary deficiencies of iron and zinc cause human malnutrition that can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success, and our understanding of the genetic and physiological barriers to altering this trait is incomplete. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat VACUOLAR IRON TRANSPORTER gene TaVIT2-D with constitutive expression of the rice (Oryza sativa) NICOTIANAMINE SYNTHASE gene OsNAS2 significantly increases the total concentration of zinc and relocates iron to white-flour fractions. In two distinct bread wheat cultivars, we show that the so called VIT-NAS construct led to a two-fold increase in zinc in wholemeal flour, to ∼50 µg g-1. Total iron was not significantly increased, but redistribution within the grain resulted in a three-fold increase in iron in highly pure, roller-milled white flour, to ∼25 µg g-1. Interestingly, expression of OsNAS2 partially restored iron translocation to the aleurone, which is iron depleted in grain overexpressing TaVIT2 alone. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines corresponded with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. Our results provide insights into mineral translocation and distribution in wheat grain and demonstrate that the individual and combined effects of the two transgenes can enhance the nutritional quality of wheat beyond what is possible by conventional breeding.

Figure 1

Expression of TaVIT2-D and OsNAS2 in the VIT-NAS lines. A, Diagram of the T-DNA construct (not to scale): HMWG, HIGH-MOLECULAR-WEIGHT GLUTENIN-D1-1 promoter; TaVIT2-D, wheat (T. aestivum) VACUOLAR IRON TRANSPORTER2-D gene; nosT (bacterial) nopaline synthase terminator; ZmUBI1, maize (Zea mays) UBIQUITIN promoter; OsNAS2, rice (O. sativa) NICOTIANAMINE SYNTHASE 2 gene; 35S, Cauliflower Mosaic Virus 35S promoter; HYG, hygromycin resistance gene; 35ST, Cauliflower Mosaic Virus 35S terminator; LB, left border. B and C, RT–qPCR expression of (B) TaVIT2-D using primers specific for the transgene and (C) OsNAS2 in grain (left) and flag leaf (right) tissue at 21 days postanthesis. Expression levels were calculated relative to TaACTIN for null transformant (A1) and transgenic (A10, B4, B12, B14, and B71) lines, and represented on a base-10 log scale. Error bars represent the standard error of three biological replicates for each line.

Figure 2

The VIT-NAS construct does not affect plant growth. A, Representative individual wheat plants of the VIT-NAS T₃ generation at 5 days after anthesis. B, Plant growth parameters including harvest index, height, thousand grain weight, and grain yield per plant in the null transformant (A1; light blue), VIT-NAS (A10, B4, B12, B14, B71; dark blue), and TaVIT2 (green) lines (*P < 0.05, Dunnett’s test against A1). Error bars are the standard error of five biological replicates.

Figure 3

The VIT-NAS lines have increased iron and zinc concentrations in flour fractions. A and B, Iron and zinc concentrations measured by ICP-OES in white flour (A) and wholemeal flour (B) of the null transformant (A1, light blue), VIT-NAS (A10, B4, B12, B14, and B71; dark blue), and TaVIT2 (green) lines in the homozygous T₃ generation. Error bars represent the standard error of five biological replicates. Student’s t test against the null (A1); *P < 0.05, **P < 0.01, ***P < 0.001. C, Iron and zinc concentrations in roller-milled fractions of grain from a null transformant (light blue) and VIT-NAS (B71, dark blue). The different flour fractions are: (white flour) B1, first break; B2, second break; R1, first reduction; R2, second reduction; (bran) FB1, first FB; FB2, second FB. The dashed line represents the minimum requirement for iron fortification in white flour in the UK (16.5 µg g⁻¹). Error bars represent the standard error of three technical replicates. Student’s t test between null and B71 for each fraction; *P < 0.05, **P < 0.01, ***P < 0.001. D, Iron and zinc concentrations in flag leaf tissue 21-day postanthesis. Error bars represent the standard error of three to four biological replicates. Student’s t test against the null (A1); *P < 0.05.

Figure 4

TaVIT2 and OsNAS2 affect the distribution of iron in the grain. A, Cross sections of mature grains stained for iron (blue) using the Perls’ method. Scale bars are 1 mm. B, Thin sections (1 µm) of immature grains 21 days after anthesis stained for iron (black in monochrome images) using the Perls’-diaminobenzidine method. Left, cross-section through the grain; middle, detail at higher magnification of the starchy endosperm between the vascular bundle and embryo; right, detail including the aleurone tissue. The images are representative of two grains taken from two different plants from the indicated wheat lines. Perls’-diaminobenziden staining of TaVIT2 grain was previously described (Sheraz et al., 2021). Al, aleurone; MA, modified aleurone; Pc, pericarp; Sc, scutellum of the embryo; SE, starchy endosperm; V, vascular bundle (maternal tissue). White asterisk indicates a nonspecific dye precipitate; black arrows point at iron accumulation in the vacuoles of starchy endosperm cells. Scale bars as indicated.

Figure 5

The VIT-NAS lines contain higher levels of NA. NA concentration in grain of the control (A1, light blue) and VIT-NAS (A10, B4, B12, B14, and B71, dark blue) lines. NA levels are plotted against a base-10 log scale. Error bars represent the standard error of three biological replicates. **P < 0.01; Mann–Whitney test compared to the control line.