Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification

Abstract

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat (Triticum aestivum) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast (Saccharomyces cerevisiae) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley (Hordeum vulgare). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms.

Figure 1.

The wheat genome encodes two VIT paralogs with different expression patterns. A, Phylogenetic tree of VIT genes from selected plant species: At, Arabidopsis thaliana; Gm, Glycine max (soybean); Hv, Hordeum vulgare (barley); Os, Oryza sativa (rice); Sl, Solanum lycopersicum (potato); Ta, Triticum aestivum (wheat); Vv, Vitis vinifera (grape); Zm, Zea mays (maize). Numbers above or below branches represent bootstrapping values for 100 replications. B, Gene expression profiles of TaVIT1 and TaVIT2 homoeologs using RNA-seq data from expVIP. Bars indicate mean transcripts per million (TPM) ± se. Full details and metadata are given in Supplemental Table S2.

Figure 2.

TaVIT2 facilitates iron and manganese transport. A to C, Yeast complementation assays of TaVIT1 and TaVIT2 in Δccc1 (A), Δpmr1 (B), and Δzrc1 (C) compared with yeast that is wild type (WT) for these three genes. The yeast (Sc) CCC1, PMR1, and ZRC1 genes were used as positive controls. Cells were spotted in a 4-fold dilution series and grown for 2 to 3 d on plates with or without 7.5 mm FeSO₄ (Δccc1), 2 mm MnCl₂ (Δpmr1), or 5 mm ZnSO₄ (Δzrc1). D, Immunoblots of total and vacuolar protein fractions from yeast cells expressing hemagglutinin (HA)-tagged TaVIT1 or TaVIT2. The HA tag did not inhibit the function of TaVIT2, as it was able to complement Δccc1 yeast (data not shown). Vhp1 was used as a vacuolar marker, and the absence of actin shows the purity of the vacuolar fraction.

Figure 3.

Expression of TaVIT2 in cisgenic lines. A, Diagram of the transfer DNA construct: LB, left border; 35S, cauliflower mosaic virus 35S promoter; HYG, hygromycin resistance gene; nosT, nos terminator; HMW-GLU prom, high-molecular-weight glutenin-D1-1 promoter; TaVIT2, wheat VIT2-D gene; RB, right border; nt, nucleotides. B, Relative expression levels of TaVIT2 in developing grains at 10 d post-anthesis as determined by quantitative real-time PCR and normalized to housekeeping gene Traes_4AL_8CEA69D2F. Plant identification numbers and copy numbers of the HMW-TaVIT2 gene are given below the bars. Bars indicate means ± se of three independent biological replicates.

Figure 4.

Perls’ Prussian Blue staining for iron in grains transformed with HMW-TaVIT2. Grains from T0 wheat plants were dissected longitudinally (left) or transversely (right). al, Aleurone; em, embryo; es, endosperm; gr, groove; s, scutellum; sdc, seed coat. The transgene copy numbers and line numbers are indicated at left. Bars = 1 mm.

Figure 5.

Iron and phytate content of flour milled from HMW-TaVIT2 wheat lines. A, Iron concentrations in white and whole-meal flour from three control and six HMW-TaVIT2 lines. Bars represent means of two technical replicates ± sd. White flour from HMW-TaVIT2 lines has significantly more iron than that from control lines (n = 3–4, P < 0.001; for all data, see Supplemental Table S3). The dotted line at 16.5 μg g⁻¹ iron indicates the minimum requirement for wheat flour sold in the United Kingdom. B, Phytate content of white and whole-meal flour of control and HMW-TaVIT2-expressing wheat. Bars represent means of two biological replicates ± sd. C, Molar ratio of iron to phytate in control and HMW-TaVIT2-expressing lines. Bars represent means of two biological replicates ± sd.

Figure 6.

Growth parameters of HMW-TaVIT2 wheat. The number of tillers (A) and seed output (B) of T0 wheat plants with indicated HMW-TaVIT2 copy numbers are shown. Bars indicate means ± se of the following numbers of biological replicates: zero gene copies, n = 9; one gene copy, n = 10; two to 16 gene copies, n = 9; 20 or more gene copies, n = 6. Further details are given in Supplemental Table S5. The asterisk indicates a significant difference from the negative control (one-way ANOVA with Tukey’s posthoc test: *, P < 0.05).

Figure 7.

Endosperm-specific overexpression of TaVIT2 in barley. The TaVIT2-5DL gene from wheat under the control of the wheat HMW-GLU-1D-1 promoter (for full details, see Fig. 3A) was transformed into barley cv Golden Promise. Positive transformants were selected by hygromycin. A, Mature barley T1 grains of a control plant and two transgenic lines stained with Perls’ Prussian Blue for iron. B, Element analysis in white and whole-meal flours from a control and two HMW-TaVIT2-overexpressing barley plants. The values are means of two technical replicates ± sd.