Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

Effect of Rice GDP-L-Galactose Phosphorylase Constitutive Overexpression on Ascorbate Concentration, Stress Tolerance, and Iron Bioavailability in Rice.

Ascorbate (vitamin C) is an essential multifunctional molecule for both plants and mammals. In plants, ascorbate is the most abundant water-soluble antioxidant that supports stress tolerance. In humans, ascorbate is an essential micronutrient and promotes iron (Fe) absorption in the gut. Engineering crops with increased ascorbate levels have the potential to improve both crop stress tolerance and human health. Here, rice (Oryza sativa L.) plants were engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase coding sequence (35S-OsGGP), which encodes the rate-limiting enzymatic step of the L-galactose pathway. Ascorbate concentrations were negligible in both null segregant (NS) and 35S-OsGGP brown rice (BR, unpolished grain), but significantly increased in 35S-OsGGP germinated brown rice (GBR) relative to NS. Foliar ascorbate concentrations were significantly increased in 35S-OsGGP plants in the vegetative growth phase relative to NS, but significantly reduced at the reproductive growth phase and were associated with reduced OsGGP transcript levels. The 35S-OsGGP plants did not display altered salt tolerance at the vegetative growth phase despite having elevated ascorbate concentrations. Ascorbate concentrations were positively correlated with ferritin concentrations in Caco-2 cells - an accurate predictor of Fe bioavailability in human digestion - exposed to in vitro digests of NS and 35S-OsGGP BR and GBR samples.

Manipulation of Ascorbate Biosynthetic, Recycling, and Regulatory Pathways for Improved Abiotic Stress Tolerance in Plants.

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.

Nicotianamine-chelated iron positively affects iron status, intestinal morphology and microbial populations in vivo (Gallus gallus).

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 NA-chelated Fe on gastrointestinal health and functionality.

Genome-wide identification and characterization of the GDP-L-galactose phosphorylase gene family in bread wheat.

BACKGROUND: Ascorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway-the dominant ascorbate biosynthetic pathway in plants-and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5'-untranslated region (5'UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans. RESULTS: Bread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5'UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta. CONCLUSIONS: We have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability.

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 (OsNAS2) 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-OsNAS2 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-OsNAS2 grain and positively correlated with NA and DMA concentrations.

Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach.

Iron deficiency currently affects over two billion people worldwide despite significant advances in technology and society aimed at mitigating this global health problem. Biofortification of food staples with iron (Fe) represents a sustainable approach for alleviating human Fe deficiency in developing countries, however, biofortification efforts have focused extensively on cereal staples while pulses have been largely overlooked. In this study we describe a genetic engineering (GE) approach to biofortify the pulse crop, chickpea (Cicer arietinum L.), with Fe using a combination of the chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes which function in Fe transport and storage, respectively. This study consists of three main components: (1) the establishment for baseline Fe concentration of existing germplam, (2) the isolation and study of expression pattern of the novel CaNAS2 gene, and (3) the generation of GE chickpea overexpressing the CaNAS2 and GmFER genes. Seed of six commercial chickpea cultivars was collected from four different field locations in Australia and assessed for seed Fe concentration. The results revealed little difference between the cultivars assessed, and that chickpea seed Fe was negatively affected where soil Fe bioavailability is low. The desi cultivar HatTrick was then selected for further study. From it, the CaNAS2 gene was cloned and its expression in different tissues examined. The gene was found to be expressed in multiple vegetative tissues under Fe-sufficient conditions, suggesting that it may play a housekeeping role in systemic translocation of Fe. Two GE chickpea events were then generated and the overexpression of the CaNAS2 and GmFER transgenes confirmed. Analysis of nicotianamine (NA) and Fe levels in the GE seeds revealed that NA was nearly doubled compared to the null control while Fe concentration was not changed. Increased NA content in chickpea seed is likely to translate into increased Fe bioavailability and may thus overcome the effect of the bioavailability inhibitors found in pulses; however, further study is required to confirm this. This is the first known example of GE Fe biofortified chickpea; information gleaned from this study can feed into future pulse biofortification work to help alleviate global Fe deficiency.

Characterisation of the nicotianamine aminotransferase and deoxymugineic acid synthase genes essential to Strategy II iron uptake in bread wheat (Triticum aestivum L.).

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.

Association of Increased Grain Iron and Zinc Concentrations with Agro-morphological Traits of Biofortified Rice.

Biofortification of rice (Oryza sativa L.) with micronutrients is widely recognized as a sustainable strategy to alleviate human iron (Fe) and zinc (Zn) deficiencies in developing countries where rice is the staple food. Constitutive overexpression of the rice nicotianamine synthase (OsNAS) genes has been successfully implemented to increase Fe and Zn concentrations in unpolished and polished rice grain. Intensive research is now needed to couple this high-micronutrient trait with high grain yields. We investigated associations of increased grain Fe and Zn concentrations with agro-morphological traits of backcross twice second filial (BC2F2) transgenic progeny carrying OsNAS1 or OsNAS2 overexpression constructs under indica/japonica and japonica/japonica genetic backgrounds. Thirteen agro-morphological traits were evaluated in BC2F2 transgenic progeny grown under hydroponic conditions. Concentrations of eight mineral nutrients (Fe, Zn, copper, manganese, calcium, magnesium, potassium, and phosphorus) in roots, stems/sheaths, non-flag leaves, flag leaves, panicles, and grain were also determined. A distance-based linear model (DistLM) was utilized to extract plant tissue nutrient predictors accounting for the largest variation in agro-morphological traits differing between transgenic and non-transgenic progeny. Overall, the BC2F2 transgenic progeny contained up to 148% higher Fe and 336% higher Zn concentrations in unpolished grain compared to non-transgenic progeny. However, unpolished grain concentrations surpassing 23 µg Fe g-1 and 40 µg Zn g-1 in BC2F2indica/japonica progeny, and 36 µg Fe g-1 and 56 µg Zn g1 in BC2F2japonica/japonica progeny, were associated with significant reductions in grain yield. DistLM analyses identified grain-Zn and panicle-magnesium as the primary nutrient predictors associated with grain yield reductions in the indica/japonica and japonica/japonica background, respectively. We subsequently produced polished grain from high-yield BC2F2 transgenic progeny carrying either the OsNAS1 or OsNAS2 overexpression constructs. The OsNAS2 overexpressing progeny had higher percentages of Fe and Zn in polished rice grain compared to the OsNAS1 overexpressing progeny. Results from this study demonstrate that genetic background has a major effect on the development of Fe and Zn biofortified rice. Moreover, our study shows that high-yielding rice lines with Fe and Zn biofortified polished grain can be developed by OsNAS2 overexpression and monitoring for Zn overaccumulation in the grain.