Summary Bread wheat ( Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression ( CE ) of the rice ( Oryza sativa L.) nicotianamine synthase 2 ( Os NAS 2 ) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine ( NA ) and 2′‐deoxymugineic acid ( DMA ) – that play key roles in metal transport and nutrition. The CE ‐ Os NAS 2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy ( XFM ) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE ‐ Os NAS 2 grain and positively correlated with NA and DMA concentrations.

Improved mapping, multi-environment quantitative trait loci (QTL) analysis and dissection of allelic effects were used to define a QTL associated with grain yield, thousand grain weight and early vigour on chromosome 3BL of bread wheat (Triticum aestivum L.) under abiotic stresses. The QTL had pleiotropic effects and showed QTL x environment interactions across 21 diverse environments in Australia and Mexico. The occurrence and the severity of water deficit combined with high temperatures during the growing season affected the responsiveness of this QTL, resulting in a reversal in the direction of allelic effects. The influence of this QTL can be substantial, with the allele from one parent (RAC875) increasing grain yield by up to 12.5 % (particularly in environments where both heat and drought stress occurred) and the allele from the other parent (Kukri) increasing grain yield by up to 9 % in favourable environments. With the application of additional markers and the genotyping of additional recombinant inbred lines, the genetic map in the QTL region was refined to provide a basis for future positional cloning.

Wheat flour iron (Fe) fortification is mandatory in 75 countries worldwide yet many Fe fortificants, such as Fe-ethylenediaminetetraacetate (EDTA), result in unwanted sensory properties and/or gastrointestinal dysfunction and dysbiosis. Nicotianamine (NA) is a natural chelator of Fe, zinc (Zn) and other metals in higher plants and NA-chelated Fe is highly bioavailable in vitro. In graminaceous plants NA serves as the biosynthetic precursor to 2′-deoxymugineic acid (DMA), a related Fe chelator and enhancer of Fe bioavailability, and increased NA/DMA biosynthesis has proved an effective Fe biofortification strategy in several cereal crops. Here we utilized the chicken (Gallus gallus) model to investigate impacts of NA-chelated Fe on Fe status and gastrointestinal health when delivered to chickens through intraamniotic administration (short-term exposure) or over a period of six weeks as part of a biofortified wheat diet containing increased NA, Fe, Zn and DMA (long-term exposure). Striking similarities in host Fe status, intestinal functionality and gut microbiome were observed between the short-term and long-term treatments, suggesting that the effects were largely if not entirely due to consumption of NA-chelated Fe. These results provide strong support for wheat with increased NA-chelated Fe as an effective biofortification strategy and uncover novel impacts of NAchelated Fe on gastrointestinal health and functionality. Iron (Fe) supplementation and fortification are the two most widely used strategies to combat human Fe deficiencies that affect over 2 billion people worldwide 1-3. Iron supplementation involves large dose delivery of highly absorbable (bioavailable) Fe to humans and is effective in treating severe cases of Fe deficiency anemia 2,4,5. Iron fortification involves low dose delivery of bioavailable Fe fortificants to food products during manufacture (or point-of-use) and is an effective population-based strategy to boost Fe intakes. Iron fortification of wheat flour is now mandatory in 75 countries worldwide (Flour Fortification Initiative; https://fortificationdata.org/), however, the tendency of Fe fortificants such as ferrous sulfate (FeSO 4) to oxidize and cause undesired organoleptic and sensory properties pose significant challenges 6,7. Almost 90% of countries utilize fortificants with poor bioavailability or fortify at sub-optimal concentrations, although recent evidence suggests that Fe fortification can effectively reduce symptoms of Fe-deficiency anemia when correctly implemented 8-10. Iron chelated by ethylenediaminetetraacetate (EDTA) is a commonly recommended fortificant for cereal flour to minimize sensory alterations while providing Fe in a bioavailable form 3,6,8,11,12. Fortificants that utilize micro-and/or nanoencapsulation can further improve bioavailability 12-14 although the cost of using appropriately chelated and/or encapsulated Fe fortificants ($2 USD per ton to fortify wheat flour with EDTA-chelated Fe alone), and the requirement for centralized cereal processing and indust...

SummaryNicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.

Abiotic stresses, such as drought, salinity, and extreme temperatures, are major limiting factors in global crop productivity and are predicted to be exacerbated by climate change. The overproduction of reactive oxygen species (ROS) is a common consequence of many abiotic stresses. Ascorbate, also known as vitamin C, is the most abundant water-soluble antioxidant in plant cells and can combat oxidative stress directly as a ROS scavenger, or through the ascorbate–glutathione cycle—a major antioxidant system in plant cells. Engineering crops with enhanced ascorbate concentrations therefore has the potential to promote broad abiotic stress tolerance. Three distinct strategies have been utilized to increase ascorbate concentrations in plants: (i) increased biosynthesis, (ii) enhanced recycling, or (iii) modulating regulatory factors. Here, we review the genetic pathways underlying ascorbate biosynthesis, recycling, and regulation in plants, including a summary of all metabolic engineering strategies utilized to date to increase ascorbate concentrations in model and crop species. We then highlight transgene-free strategies utilizing genome editing tools to increase ascorbate concentrations in crops, such as editing the highly conserved upstream open reading frame that controls translation of the GDP-L-galactose phosphorylase gene.

Iron (Fe) uptake in graminaceous plant species occurs via the release and uptake of Fe-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the MAs biosynthetic pathway, nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) enzymes catalyse the formation of 2’-deoxymugineic acid (DMA) from nicotianamine (NA). Here we describe the identification and characterisation of six TaNAAT and three TaDMAS1 genes in bread wheat (Triticum aestivum L.). The coding sequences of all six TaNAAT homeologs consist of seven exons with ≥88.0% nucleotide sequence identity and most sequence variation present in the first exon. The coding sequences of the three TaDMAS1 homeologs consist of three exons with ≥97.8% nucleotide sequence identity. Phylogenetic analysis revealed that the TaNAAT and TaDMAS1 proteins are most closely related to the HvNAAT and HvDMAS1 proteins of barley and that there are two distinct groups of TaNAAT proteins—TaNAAT1 and TaNAAT2 –that correspond to the HvNAATA and HvNAATB proteins, respectively. Quantitative reverse transcription-PCR analysis revealed that the TaNAAT2 genes are expressed at highest levels in anther tissues whilst the TaNAAT1 and TaDMAS1 genes are expressed at highest levels in root tissues of bread wheat. Furthermore, the TaNAAT1, TaNAAT2 and TaDMAS1 genes were differentially regulated by plant Fe status and their expression was significantly upregulated in root tissues from day five onwards during a seven-day Fe deficiency treatment. The identification and characterization of the TaNAAT1, TaNAAT2 and TaDMAS1 genes provides a valuable genetic resource for improving bread wheat growth on Fe deficient soils and enhancing grain Fe nutrition.

ORCID IDs: 0000-0002-7600-9592 (B.P.); 0000-0003-3851-8617 (J.B.); 0000-0001-9494-400X (P.L.); 0000-0002-7077-4103 (D.F.).Yield is subject to strong genotype-by-environment (G 3 E) interactions in the field, especially under abiotic constraints such as soil water deficit (drought [D]) and high temperature (heat [H]). Since environmental conditions show strong fluctuations during the whole crop cycle, geneticists usually do not consider environmental measures as quantitative variables but rather as factors in multienvironment analyses. Based on 11 experiments in a field platform with contrasting temperature and soil water deficit, we determined the periods of sensitivity to drought and heat constraints in wheat (Triticum aestivum) and determined the average sensitivities for major yield components. G 3 E interactions were separated into their underlying components, constitutive genotypic effect (G), G 3 D, G 3 H, and G 3 H 3 D, and were analyzed for two genotypes, highlighting contrasting responses to heat and drought constraints. We then tested the constitutive and responsive behaviors of two strong quantitative trait loci (QTLs) associated previously with yield components. This analysis confirmed the constitutive effect of the chromosome 1B QTL and explained the G 3 E interaction of the chromosome 3B QTL by a benefit of one allele when temperature rises. In addition to the method itself, which can be applied to other data sets and populations, this study will support the cloning of a major yield QTL on chromosome 3B that is highly dependent on environmental conditions and for which the climatic interaction is now quantified.

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