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

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.

Subcellular dynamics studies of iron reveal how tissue-specific distribution patterns are established in developing wheat grains.

Understanding the mechanisms of iron trafficking in plants is key to enhancing the nutritional quality of crops. Because it is difficult to image iron in transit, we currently have an incomplete picture of the route(s) of iron translocation in developing seeds and how the tissue-specific distribution is established. We have used a novel approach, combining iron-57 (57 Fe) isotope labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), to visualize iron translocation between tissues and within cells in immature wheat grain, Triticum aestivum. This enabled us to track the main route of iron transport from maternal tissues to the embryo through the different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, with confirmation provided by histological staining and transmission electron microscopy energy dispersive X-ray spectroscopy (TEM-EDS). Almost all iron in both control and transgenic grains was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Furthermore, a new type of iron body, highly enriched in 57 Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids. Correlation of the 57 Fe enrichment profiles obtained by NanoSIMS with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification strategies.

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling.

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake.

Iron Biofortification of Staple Crops: Lessons and Challenges in Plant Genetics.

Plants are the ultimate source of iron in our diet, either directly as staple crops and vegetables or indirectly via animal fodder. Increasing the iron concentration of edible parts of plants, known as biofortification, is seen as a sustainable approach to alleviate iron deficiency which is a major global health issue. Advances in sequencing and gene technology are accelerating both forward and reverse genetic approaches. In this review, we summarize recent progress in iron biofortification using conventional plant breeding or transgenics. Interestingly, some of the gene targets already used for transgenic approaches are also identified as genetic factors for high iron in genome-wide association studies. Several quantitative trait loci and transgenes increase both iron and zinc, due to overlap in transporters and chelators for these two mineral micronutrients. Research efforts are predominantly aimed at increasing the total concentration of iron but enhancing its bioavailability is also addressed. In particular, increased biosynthesis of the metal chelator nicotianamine increases iron and zinc levels and improves bioavailability. The achievements to date are very promising in being able to provide sufficient iron in diets with less reliance on meat to feed a growing world population.

Iron homeostasis in plants - a brief overview.

Iron plays a crucial role in biochemistry and is an essential micronutrient for plants and humans alike. Although plentiful in the Earth's crust it is not usually found in a form readily accessible for plants to use. They must therefore sense and interact with their environment, and have evolved two different molecular strategies to take up iron in the root. Once inside, iron is complexed with chelators and distributed to sink tissues where it is used predominantly in the production of enzyme cofactors or components of electron transport chains. The processes of iron uptake, distribution and metabolism are overseen by tight regulatory mechanisms, at the transcriptional and post-transcriptional level, to avoid iron concentrations building to toxic excess. Iron is also loaded into seeds, where it is stored in vacuoles or in ferritin. This is important for human nutrition as seeds form the edible parts of many crop species. As such, increasing iron in seeds and other tissues is a major goal for biofortification efforts by both traditional breeding and biotechnological approaches.

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

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.

Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops.

Wheat, like many other staple cereals, contains low levels of the essential micronutrients iron and zinc. Up to two billion people worldwide suffer from iron and zinc deficiencies, particularly in regions with predominantly cereal-based diets. Although wheat flour is commonly fortified during processing, an attractive and more sustainable solution is biofortification, which requires developing new varieties of wheat with inherently higher iron and zinc content in their grains. Until now most studies aimed at increasing iron and zinc content in wheat grains have focused on discovering natural variation in progenitor or related species. However, recent developments in genomics and transformation have led to a step change in targeted research on wheat at a molecular level. We discuss promising approaches to improve iron and zinc content in wheat using knowledge gained in model grasses. We explore how the latest resources developed in wheat, including sequenced genomes and mutant populations, can be exploited for biofortification. We also highlight the key research and practical challenges that remain in improving iron and zinc content in wheat.

Knockout of multiple Arabidopsis cation/H(+) exchangers suggests isoform-specific roles in metal stress response, germination and seed mineral nutrition.

Cation/H(+) exchangers encoded by CAX genes play an important role in the vacuolar accumulation of metals including Ca(2+) and Mn(2+). Arabidopsis thaliana CAX1 and CAX3 have been previously shown to differ phylogenetically from CAX2 but the physiological roles of these different transporters are still unclear. To examine the functions and the potential of redundancy between these three cation transporters, cax1/cax2 and cax2/cax3 double knockout mutants were generated and compared with wild type and cax single knockouts. These double mutants had equivalent metal stress responses to single cax mutants. Both cax1 and cax1/cax2 had increased tolerance to Mg stress, while cax2 and cax2/cax3 both had increased sensitivity to Mn stress. The cax1/cax2 and cax2/cax3 mutants did not exhibit the deleterious developmental phenotypes previously seen with the cax1/cax3 mutant. However, these new double mutants did show alterations in seed germination, specifically a delay in germination time. These alterations correlated with changes in nutrient content within the seeds of the mutants, particularly the cax1/cax2 mutant which had significantly higher seed content of Ca and Mn. This study indicates that the presence of these Arabidopsis CAX transporters is important for normal germination and infers a role for CAX proteins in metal homeostasis within the seed.

Functional studies of split Arabidopsis Ca2+/H+ exchangers.

In plants, high capacity tonoplast cation/H(+) antiport is mediated in part by a family of cation exchanger (CAX) transporters. Functional association between CAX1 and CAX3 has previously been shown. In this study we further examine the interactions between CAX protein domains through the use of nonfunctional halves of CAX transporters. We demonstrate that a protein coding for an N-terminal half of an activated variant of CAX1 (sCAX1) can associate with the C-terminal half of either CAX1 or CAX3 to form a functional transporter that may exhibit unique transport properties. Using yeast split ubiquitin, in planta bimolecular fluorescence complementation, and gel shift experiments, we demonstrate a physical interaction among the half proteins. Moreover, the half-proteins both independently localized to the same yeast endomembrane. Co-expressing variants of N- and C-terminal halves of CAX1 and CAX3 in yeast suggested that the N-terminal region mediates Ca(2+) transport, whereas the C-terminal half defines salt tolerance phenotypes. Furthermore, in yeast assays, auto-inhibited CAX1 could be differentially activated by CAX split proteins. The N-terminal half of CAX1 when co-expressed with CAX1 activated Ca(2+) transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca(2+) transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.

Comparative analysis of CAX2-like cation transporters indicates functional and regulatory diversity.

Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.