Constitutive overexpression of a nicotianamine synthase gene in bread wheat and in vivo assessment of iron and zinc bioavailability

Constitutive overexpression of a nicotianamine synthase gene in bread wheat and in vivo assessment of iron and zinc bioavailability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Constitutive overexpression of a nicotianamine synthase gene in bread wheat and in vivo assessment of iron and zinc bioavailability Elad Tako, Jacquelyn Cheng, Jesse T. Beasley, Nikolai Kolba, Cydney Jackson, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4631411/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Iron (Fe) and zinc (Zn) deficiencies affect over two billion people globally. Biofortification of bread wheat (Triticum aestivum), a crop that supplies approximately 20% of calories and protein consumed by humans worldwide, represents a sustainable strategy for increasing micronutrient intakes. We employed constitutive overexpression (OE) of an endogenous nicotianamine synthase gene (TaNAS2A) in bread wheat cultivar Gladius to increase biosynthesis of the metal-chelating molecule nicotianamine (NA). Field evaluation of three independent OE-TaNAS2A events found normal growth and consistently increased NA concentration in whole wheat flour relative to controls. Extracts prepared from whole wheat flours were functionally characterized in vivo (Gallus gallus) using the intraamniotic administration approach and alterations in markers of Fe and Zn transport, inflammation, and intestinal functionality and morphology were observed in treatment groups that received OE-TaNAS2A extracts. Biological sciences/Plant sciences Health sciences/Biomarkers Health sciences/Diseases Figures Figure 1 Figure 2 Figure 3 Introduction Iron (Fe) deficiency is the most common nutritional disorder in humans, affecting approximately two billion people including 40% of pregnant women and 42% of children below 5 years of age 1–4 . Zinc (Zn) deficiency is the second most common nutritional disorder in humans, estimated to affect 1 billion people, or 17% of the global population 2,3,5 . Iron and Zn deficiencies can have severe consequences on human health, such as depressed immunity, hindered development, and cognitive impairment 6–9 . Mineral supplementation strategies are efficacious at alleviating severe instances of mineral deficiency, though supplementation efforts in developing regions are often unsuccessful at the population level due to inadequate infrastructure and education 10,11 . Food fortification strategies involve the addition of mineral fortificants to food during processing and are effectively implemented in over 90 countries, however fortification efforts require suitable delivery systems, processing facilities, policies, and constant funding to be effective 12,13 . Further, undesirable sensory characteristics have been associated with Fe fortification of food 13–15 . By contrast, biofortification, the process of increasing the nutritional quality of food crops through conventional plant breeding and/or modern biotechnology without sacrificing the preferences of consumers and farmers, represents a more sustainable method for alleviating mineral deficiencies in vulnerable populations 16–18 . Implementing biofortified foods does not require changes to dietary patterns for target populations and can reach rural populations that have poor access to infrastructure 19 . Previous Fe and Zn biofortification efforts in developing regions have demonstrated increases in dietary mineral intakes that are associated with a reduction in human Fe and/or Zn deficiencies 18,20–23 . The consumption of Fe- and Zn-biofortified foods can increase the colonization of beneficial bacteria in the host gut microbiome and potentially improve intestinal health 12,24–28 . Crop biofortification with Fe and/or Zn may increase the yield of staple crops under Fe- and/or Zn-deprived soil conditions 29 . Furthermore, economic analyses have demonstrated that biofortification is the most robust and cost-effective strategy for increasing dietary Fe and/or Zn intakes within vulnerable populations in comparison to dietary diversification, supplementation, or food fortification programs 10,11,30 . Crop-derived bioactive compounds have been shown to improve mineral bioavailability, possess anti-inflammatory properties, and positively modulate the gut microbiome 12,31–34 . Nicotianamine (NA), a non-protein amino acid-derived plant metabolite, is a naturally occurring chelator of iron (Fe), zinc (Zn), and other transition metals in higher plants and is involved in metal translocation between plant cells, tissues and organs 35,36 . Both NA and its downstream metabolite (2′-deoxymugineic acid, DMA) are major mineral chelators in white wheat flour 37 and are thought to be enhancers of in vitro and in vivo Fe bioavailability 12,38,39 . Within intestinal enterocytes (absorptive cells), Fe can be absorbed as an NA-Fe complex via the proton-coupled transporter 1 (PAT1) rather than as inorganic Fe 39 . For these reasons, NA has been described as a bioactive compound that enhances in vivo Fe bioavailability, and increased biosynthesis of NA/DMA represents a promising strategy for Fe and Zn biofortification of staple crops 12,38,40–42 . Bread wheat ( Triticum aestivum ) supplies 20% of food calories to the world’s population and is the most widely cultivated crop globally 43 . In some low-income communities, wheat accounts for up to 90% of total dietary energy intake 44 , and wheat biofortification efforts aimed at increasing mineral intakes may improve mineral status and reduce the rates of morbidity and mortality in these regions 30,45,46 . We recently assessed in vivo ( Gallus gallus ) Fe bioavailability of biofortified white wheat flour, where NA and DMA concentrations were increased via constitutive expression of the rice ( Oryza sativa ) nicotianamine synthase 2 ( OsNAS2 ) gene 12,40 . Short-term exposure (intraamniotic administration) to OsNAS2 biofortified white wheat flour extract resulted in improvements in intestinal morphology but not alterations in mineral transport gene expression, and long-term exposure (six-week feeding trial) to biofortified white wheat flour resulted in significant positive changes in host Fe status, brush border membrane functionality and development, and gut microbiota structure and function 12 . Here we describe constitutive overexpression (OE) of an endogenous bread wheat nicotianamine synthase 2A ( TaNAS2A ) gene as a potential strategy for generating Fe and Zn biofortified wheat and to assess in vivo Fe and Zn bioavailability within grain produced from field-grown OE- TaNAS2A and null segregant (NS) control wheat 40,47 . We examine the impact of OE- TaNAS2A and NS whole wheat flour extracts on mineral (Fe and Zn) status, inflammatory status, and intestinal functionality and morphology, utilizing the G. gallus model intraamniotic administration approach (embryonic stage, short-term exposure). The G. gallus system is sensitive to minor modulations in dietary mineral concentration/content and is therefore frequently used to model human dietary mineral bioavailability and absorption 24,48–50 . The intraamniotic administration method involves injecting extracts into the amniotic fluid at day 17, which are entirely consumed by the embryo prior to hatch, and is widely used to assess the effect of biofortified foods on mineral transport and intestinal functionality and morphology 51–54 . Furthermore, humans and G. gallus share > 85% gene homology between intestinal brush border membrane proteins involved in mineral transport, further highlighting the relevance of the G. gallus in assessing mineral bioavailability 55 . The first objective of this study was to investigate the effect of overexpressing an endogenous TaNAS2A gene on field-grown bread wheat with respect to phenotype, yield, and grain nutritional composition. The second objective was to assess the effect of intraamniotic administration of OE- TaNAS2A whole wheat flour extracts on biomarkers of Fe and Zn bioavailability utilizing the G. gallus model. Results Plant production and field evaluation Bread wheat cultivar (cv.) Gladius transformants (OE- TaNAS2A wheats) were generated through Agrobacterium tumefaciens transformation of a T-DNA construct ( Supplementary Figure S1 ) containing the TaNAS2A gene under regulatory control of the maize ubiquitin 1 promoter (UBI-1) (Fig. 1 A). Three independent OE- TaNAS2A transformants, pUbi::TaNAS2A 1T, 2T, and 3T (hereafter referred to as OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3, respectively) and their respective null segregants (NS), pUbi::TaNAS2A 1N, 2N, and 3N (hereafter referred to as NS-1, NS-2, and NS-3, respectively, Table 1 ) were evaluated under confined field trial conditions in Victoria, Australia to assess agronomic performance. Figure 1 B shows a representative image of replicated 1m 2 plots of the OE- TaNAS2A wheat plants and Fig. 1 C displays a map of the randomized block design. Table 1 . Bread wheat genotypes, description, and reference name within this paper. TaNAS2A : wheat nicotianamine synthase 2A gene; NS, null segregant; OE, overexpression. Wheat genotype Wheat description Wheat reference name within this paper pUbi::TaNAS2A 1N Null segregant NS-1 pUbi::TaNAS2A 1T Overexpression of TaNAS2A OE-TaNAS2-1 pUbi::TaNAS2A 2N Null segregant NS-2 pUbi::TaNAS2A 2T Overexpression of TaNAS2A OE-TaNAS2-2 pUbi::TaNAS2A 3N Null segregant NS-3 pUbi::TaNAS2A 3T Overexpression of TaNAS2A OE-TaNAS2-3 Agromorphological data gathered from field evaluation of the six wheat genotypes is summarized in Table 2 . No significant differences in plant height or tiller number were detected between genotypes. Average spikelet number was significantly increased ( p < 0.05) in OE-TaNAS2-2 relative to NS-2 and plot yield (g per m 2 ) was significantly increased ( p < 0.05) in OE-TaNAS2-1 and NS-3 relative to NS-1 (Table 2 ). Table 2 Agromorphological data of three OE- TaNAS2A genotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, and NS-3) . a-b Per parameter, wheat varieties not indicated by the same letter are significantly different ( p < 0.05) according to one-way ANOVA with post-hoc Duncan test. * Per parameter, each OE- TaNAS2A genotype indicated is significantly different ( p < 0.05) relative to its respective null segregant as determined by Student’s t-test. Wheat genotype Plant height (cm) Tiller number Average spikelet number Yield (g per m 2 ) NS-1 95.67 ± 0.88 ᵃ 106.7 ± 16.71 ᵃ 14.03 ± 0.03 ᵃᵇ 1091.7 ± 120.35 ᵇ OE-TaNAS2-1 95.33 ± 2.91 ᵃ 123.0 ± 3.51 ᵃ 13.87 ± 0.37 ᵃᵇ 1452.1 ± 91.21 ᵃ , * NS-2 95.67 ± 2.60 ᵃ 109.0 ± 7.81 ᵃ 13.60 ± 0.15 ᵇ 1256.4 ± 55.54 ᵃᵇ OE-TaNAS2-2 93.00 ± 0.58 ᵃ 120.0 ± 7.37 ᵃ 14.97 ± 0.33 ᵃ , * 1278.1 ± 81.46 ᵃᵇ NS-3 90.00 ± 2.89 ᵃ 110.0 ± 3.79 ᵃ 14.33 ± 0.35 ᵃᵇ 1444.6 ± 16.06 ᵃ OE-TaNAS2-3 93.00 ± 1.00 ᵃ 106.3 ± 9.39 ᵃ 13.43 ± 0.12 ᵇ 1272.5 ± 75.79 ᵃᵇ Nicotianamine, DMA, Fe, and Zn concentrations in OE- TaNAS2A whole wheat flours Table 3 Nicotianamine (NA) and 2’-deoxymugineic acid (DMA) concentrations in whole wheat flours of three OE- TaNAS2A genotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, and NS-3) . a-b Per parameter, genotypes not indicated by the same letter are significantly different ( p < 0.05) according to one-way ANOVA with post-hoc Duncan test. * Per parameter, each OE- TaNAS2A genotype indicated is significantly different ( p < 0.05) relative to its respective null segregant as determined by Student’s t-test. Wheat genotype NA (nmol/g) ∆NA versus null segregant (nmol/g) DMA (nmol/g) ∆DMA versus null segregant (nmol/g) NS-1 15.07 ± 0.99 ᵇ - 36.08 ± 0.20 ᵇ - OE-TaNAS2-1 22.24 ± 0.76 ᵃᵇ , * 7.17 37.55 ± 0.13 ᵃᵇ , * 1.47 NS-2 20.94 ± 0.60 ᵃᵇ , - 36.97 ± 0.29 ᵃᵇ , - OE-TaNAS2-2 26.65 ± 1.31 ᵃ , * 5.71 38.27 ± 0.29 ᵃ , * 1.29 NS-3 18.39 ± 1.44 ᵇ , ᴮ - 37.00 ± 0.58 ᵃᵇ - OE-TaNAS2-3 27.24 ± 1.25 ᵃ , * 8.84 38.10 ± 0.28 ᵃ 1.10 The concentrations of nicotianamine (NA) and 2’-deoxymugineic acid (DMA) in whole wheat flours of the six genotypes are summarized in Table 3 . Whole wheat flour NA concentrations were significantly increased ( p < 0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to NS-1 and NS-3, and in all OE- TaNAS2A wheats relative to their respective NS controls (Table 3 ). Whole wheat flour DMA concentrations were significantly increased ( p < 0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to NS-1, and in the OE-TaNAS2-1 and OE-TaNAS2-2 wheats relative to their respective NS controls (NS-1 and NS-2, respectively). Table 4 Iron (Fe) and zinc (Zn) concentrations in whole wheat flours of three OE- TaNAS2A genotypes (OE-TaNAS2-1, OE-TaNAS2-2, OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, NS-3). Values represent mean ± SEM of three technical replicates. a, b Per parameter, genotypes not indicated by the same letter are significantly different ( p < 0.05) as determined by one-way ANOVA and post-hoc Duncan test. NS, null segregant; OE, overexpression; NA, nicotianamine; DMA, 2′-deoxymugineic acid. Wheat genotype Fe (mg/kg) ∆Fe versus null segregant (mg/kg) Zn (mg/kg) ∆Zn versus null segregant (mg/kg) NS-1 33.81 ± 1.38 ᵃᵇ - 39.26 ± 5.35 ᵃ - OE-TaNAS2-1 35.85 ± 1.62 ᵃ 2.04 46.60 ± 11.01 ᵃ 7.34 NS-2 30.63 ± 0.54 ᵇ - 37.74 ± 2.05 ᵃ - OE-TaNAS2-2 34.17 ± 1.27 ᵃᵇ 3.54 44.38 ± 4.18 ᵃ 6.64 NS-3 30.42 ± 1.42 ᵇ - 34.07 ± 4.05 ᵃ - OE-TaNAS2-3 33.72 ± 1.74 ᵃᵇ 3.31 47.64 ± 3.82 ᵃ 13.57 The concentrations of iron (Fe) and zinc (Zn) in whole wheat flours of the six genotypes are summarized in Table 4 . Whole wheat flour Fe concentrations were significantly increased ( p < 0.05) in OE-TaNAS2-1 relative to NS-2 and NS-3. In each OE- TaNAS2A wheat relative to its respective NS control, there was a trend of increased whole wheat flour Fe concentration. While whole wheat flour Zn concentrations between all wheat genotypes were not significantly different, there was a trend of increased Zn concentration between each OE- TaNAS2A wheat relative to its respective NS control. Fe and Zn concentrations in the whole wheat flour water extracts from all six genotypes are summarized in Supplementary Table S2 . Effects of OE- TaNAS2A whole wheat flour extracts on body weight and pectoral glycogen concentration Table 5 Effect of intraamniotic administration of OE- TaNAS2A wheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on chick ( Gallus gallus ) body weight and pectoral glycogen concentration on day of hatch (Day 21) . Values are means ± SEM, n = 3–6. a-c Treatment groups not indicated by the same letter in the same column are significantly different ( p < 0.05) according to one-way ANOVA with post-hoc Duncan test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. Group Bodyweight (g) Pectoral Glycogen (mg/mL) No injection 43.80 ± 0.98 ᵃ 0.053 ± 0.038 ᵃ H₂O injection 43.18 ± 1.15 ᵃᵇ 0.048 ± 0.012 ᵃ Zn 40.80 ± 1.26 ᵃᵇᶜ 0.088 ± 0.014 ᵃ NS-1 38.47 ± 1.09 ᵃᵇᶜ 0.123 ± 0.017 ᵃ OE-TaNAS2-1 39.44 ± 2.04 ᵇᶜ 0.147 ± 0.019 ᵃ NS-2 39.60 ± 1.39 ᵃᵇᶜ 0.143 ± 0.054 ᵃ OE-TaNAS2-2 38.40 ± 1.15 ᵃᵇᶜ 0.076 ± 0.036 ᵃ NS-3 40.54 ± 1.27 ᶜ 0.054 ± 0.019 ᵃ OE-TaNAS2-3 38.20 ± 1.45 ᵃᵇᶜ 0.049 ± 0.006 ᵃ The effects of OE- TaNAS2A whole wheat flour extracts on body weight and pectoral glycogen concentration (storage form of glucose) are summarized in Table 5 . Body weight was significantly decreased ( p < 0.05) in OE-TaNAS2-1 and NS-3 relative to No injection. No significant differences in pectoral glycogen (a measurement of energetic status in poultry) were found between treatment groups. When comparing each OE- TaNAS2A group to its respective NS control, there were no significant differences in body weight or pectoral glycogen. Supplementary Figure S2 depicts the effect OE- TaNAS2A whole wheat flour extracts on the expression of key gluconeogenesis (metabolic pathway that leads to glucose production) enzymes. No significant differences in PCK1 (phosphoenolpyruvate carboxykinase) expression were found between treatment groups. G6PC1 (glucose-6-phosphatase catalytic subunit 1) expression was significantly increased ( p < 0.05) in OE-TaNAS-2 and NS-3 compared with OE-TaNAS2-1 and NS-2. Relative gene expression of iron, zinc, BBM (brush border membrane) functionality, and inflammation-related proteins in response to OE- TaNAS2A whole wheat flour extracts Figure 2 depicts the relative expression of Fe metabolism genes in response to OE- TaNAS2A whole wheat flour extracts. DcytB (duodenal cytochrome B) expression was significantly increased ( p < 0.05) in OE-TaNAS2-2, NS-2, OE-TaNAS2-3, and NS-3 compared with OE-TaNAS2-1. DMT1 (divalent metal transporter 1) expression was significantly decreased ( p < 0.05) in Zn, NS-2, NS-3, and OE-TaNAS2-3 relative to No Injection, H₂O injection, NS-1, and OE-TaNAS2-1. Ferroportin expression was significantly increased ( p < 0.05) in NS-2, NS-3, and OE-TaNAS2-3 groups relative to No injection, H₂O injection, and NS-1. Hepcidin expression was significantly increased ( p < 0.05) in OE-TaNAS2-2 relative to H₂O injection, Zn, OE-TaNAS2-1, and NS-2. The expression of PAT1 (proton-coupled amino acid transporter 1) was significantly increased in NS-2 and NS-3 relative to No injection, H₂O injection, Zn, and NS-1. Between each OE- TaNAS2A group relative to its respective NS control group there were no significant differences in DcytB and DMT1 expression. Ferroportin and PAT1 were significantly increased ( p < 0.05) in OE-TaNAS2-1 relative to NS-1, hepcidin was significantly increased ( p < 0.05) in OE-TaNAS2-2 relative to NS-2, and PAT1 was significantly decreased ( p < 0.05) in OE-TaNAS-3 relative to NS-3. Figure 2 also depicts relative expression of Zn-related genes in response to OE- TaNAS2A whole wheat flour extracts. ZnT1 (Zn transporter 1) expression was significantly increased in NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 ( p < 0.05) relative to NS-1, Zn, No injection, and H₂O injection (Fig. 2 ). ZIP4 (Zn transport protein 4) expression was significantly decreased ( p < 0.05) in Zn, NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, OE-TaNAS2-1, No injection, and H₂O injection. ZIP9 (Zn transport protein 9) expression was significantly increased ( p < 0.05) in NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 compared with NS-1, Zn, and H₂O injection. Δ6-desaturase expression was significantly decreased in OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 relative to No injection, H₂O injection, NS-1, and OE-TaNAS2-1. Between each OE- TaNAS2A wheat group relative to its respective NS control group, there were no significant differences in ZnT1 and ZIP4 gene expression. ZIP9 was significantly increased ( p < 0.05) in OE-TaNAS2-1 relative to NS-1, Δ6-desaturase was significantly decreased ( p < 0.05) in OE-TaNAS2-2 relative to NS-2, and ZIP9 was significantly decreased ( p < 0.05) in OE-TaNAS-3 relative to NS-3. The effects of OE- TaNAS2A whole wheat flour extracts on plasma Fe and Zn content is depicted in Supplementary Table S3 . Figure 3 depicts the relative expression of inflammatory modulator and brush border membrane (BBM) functionality proteins after exposure to OE- TaNAS2A whole wheat flour extract. IL6 (interleukin 6) gene expression was significantly increased ( p < 0.05) in NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, Zn, and H₂O injection. TNF-α (tumor necrosis factor-alpha) expression was significantly increased ( p < 0.05) in the NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 groups relative to the Zn and H₂O injection controls. NF- Κ B1 (nuclear factor kappa B subunit 1) expression was significantly decreased ( p < 0.05) in NS-1 and OE-TaNAS2-1 compared with NS-3 and OE-TaNAS2-3. MUC2 (mucin 2) expression was significantly increased ( p < 0.05) in NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, Zn, and H₂O injection. Between each OE- TaNAS2A wheat group relative to its respective NS control group, there were no significant differences in TNF-α, NF- Κ B1, and MUC2 gene expression. IL6 was significantly increased ( p < 0.05) in OE-TaNAS2-1 relative to NS-1 and SI (sucrase-isomaltase) expression was significantly decreased ( p < 0.05) in OE-TaNAS2-2 relative to NS-2. Effects of OE- TaNAS2A whole wheat flour extracts on intestinal morphology: duodenal villi, depth of crypts, goblet cells, and Paneth cells Table 6 depicts the effects of OE- TaNAS2A whole wheat flour extract on duodenal villi morphology. Villus surface area was significantly increased ( p < 0.05) in OE-TaNAS2-1, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 relative to the No injection, H₂O injection, Zn, NS-1, and NS-2, and between each OE- TaNAS2A group relative to its respective NS control group. Villi goblet cell diameter and count were decreased in OE-TaNAS2-1 relative to its NS control group (NS-1). In contrast, villi goblet cell diameter and count were increased in OE-TaNAS2-2 and OE-TaNAS2-3 relative to their NS control groups (NS-2 and NS-3, respectively). Acidic goblet cells were the predominant goblet cell type within the villi and the trends in acidic villi goblet cell count were consistent with the total villi goblet cell count ( Supplementary Table S4 ). Table 6 Effect of intraamniotic administration of OE- TaNAS2A (NA-biofortified) and null segregant (control) whole wheat flour extracts on duodenal villi and villi goblet cells . Values are the means ± SEM. Three biological samples per treatment group and four segments for each biological sample were analyzed. Ten randomly selected villi were analyzed per segment and cell size measurements and counts were counted in ten randomly selected villi per segment (40 replicates per biological sample). a-e Treatment groups not indicated by the same letter in the same column are significantly different ( p < 0.05) as determined by one-way ANOVA and post-hoc Duncan test. * Per parameter, each OE- TaNAS2A group indicated is significantly different ( p < 0.05) relative to its respective null segregant group as determined by Student’s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. Treatment Group Villus Surface Area (µm²) Villi Goblet Diameter (µm) Villi Goblet Cell Count (unit) No injection 138.14 ± 3.75 ᵈ 3.46 ± 0.06 ᵃᵇ 17.92 ± 0.67 ᵉ H₂O injection 130.63 ± 4.24 ᵈᵉ 3.47 ± 0.06 ᵃ 31.16 ± 0.82 ᵇᶜ Zn 121.43 ± 3.64 ᵉ 2.72 ± 0.06 ᵈ 31.84 ± 1.00 ᵇ NS-1 144.88 ± 4.26 ᵈ 3.36 ± 0.07 ᵃᵇ 31.39 ± 0.93 ᵇᶜ OE-TaNAS2-1 182.00 ± 6.62 ᵇ , * 3.26 ± 0.07 ᵇ 27.13 ± 1.05 ᵈ , * NS-2 136.93 ± 6.24 ᵈᵉ 2.70 ± 0.08 ᵈ 28.53 ± 0.81 ᶜᵈ OE-TaNAS2-2 197.76 ± 4.80 ᵃ , * 3.00 ± 0.07 ᶜ , * 36.49 ± 1.18 ᵃ , * NS-3 163.60 ± 4.18 ᶜ 2.75 ± 0.07 ᵈ 31.56 ± 0.88 ᵇ OE-TaNAS2-3 205.34 ± 5.02 ᵃ , * 3.05 ± 0.08 ᶜ , * 34.15 ± 0.95 ᵃᵇ , * Table 7 Effect of intraamniotic administration of OE- TaNAS2A wheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on duodenal crypts, crypt goblet cells, and Paneth cells . Values are the means ± SEM. Three biological samples and four segments for each biological sample were analyzed per treatment group. Ten randomly selected crypts were analyzed per segment and cell size measurements and counts were counted in ten randomly selected crypts per segment (40 replicates per biological sample). a-e Treatment groups not indicated by the same letter in the same column are significantly different ( p < 0.05) as determined by one-way ANOVA and post-hoc Duncan test. * Per parameter, each OE- TaNAS2A group indicated is significantly different ( p < 0.05) relative to its respective null segregant group as determined by Student’s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. Treatment Group Crypt Depth (µm) Crypt Goblet Diameter (µm) Crypt Goblet Cell Count (unit) Paneth Cell Count per Crypt (unit) Paneth Cell Diameter (µm) No injection 22.02 ± 1.03 ᶜ 3.00 ± 0.07 ᵃ 7.57 ± 0.39 ᵃᵇ 1.08 ± 0.03 ᵇ 1.51 ± 0.03 ᵈ H₂O injection 21.42 ± 1.07 ᶜᵈ 2.86 ± 0.05 ᵃᵇ 8.27 ± 0.38 ᵃ 1.11 ± 0.03 ᵇ 1.44 ± 0.03 ᵈ Zn 23.77 ± 1.17 ᵇᶜ 2.44 ± 0.06 ᶜ 7.38 ± 0.39 ᵃᵇ 1.33 ± 0.05 ᵃ 1.32 ± 0.03 ᵉ NS-1 21.02 ± 0.93 ᶜᵈ 2.70 ± 0.06 ᵇ 6.15 ± 0.31 ᶜᵈ 1.07 ± 0.02 ᵇ 1.66 ± 0.03 ᶜ OE-TaNAS2-1 21.39 ± 1.11 ᶜᵈ 2.15 ± 0.05 ᵈ , * 5.74 ± 0.26 ᵈ 1.12 ± 0.03 ᵇ 1.76 ± 0.04 ᵇᶜ NS-2 18.16 ± 0.88 ᵈ 2.45 ± 0.07 ᶜ 5.60 ± 0.28 ᵈ 1.13 ± 0.03 ᵇ 1.78 ± 0.05 ᵇ OE-TaNAS2-2 26.13 ± 1.23 ᵇ , * 2.40 ± 0.05 ᶜ 6.85 ± 0.33 ᵇᶜ , * 1.06 ± 0.02 ᵇ 1.83 ± 0.04 ᵃᵇ NS-3 26.00 ± 1.31 ᵇ 2.70 ± 0.08 ᵇ 6.99 ± 0.30 ᵇᶜ 1.39 ± 0.05 ᵃ 1.42 ± 0.03 ᵈᵉ OE-TaNAS2-3 30.23 ± 1.84 ᵃ , * 2.34 ± 0.07 ᵈ , * 8.39 ± 0.32 ᵃ , * 1.07 ± 0.02 ᵇ , * 1.92 ± 0.04 ᵃ , * Table 7 depicts the effects of OE- TaNAS2A whole wheat flour extract on duodenal crypts, crypt goblet cells, and Paneth cells. Crypt depth was significantly increased ( p < 0.05) in NS-3 and OE-TaNAS2-3 relative to NS-1 and OE-TaNAS2-1, and between OE-TaNAS2-2 and OE-TaNAS2-3 relative to their respective NS control groups (NS-2 and NS-3, respectively). Crypt goblet diameter was significantly reduced ( p < 0.05) in all OE- TaNAS2A groups relative to No injection and H₂O injection controls, and between OE-TaNAS2-1 and OE-TaNAS2-3 relative to their respective NS control groups (NS-1 and NS-3, respectively). Crypt goblet cell count was significantly increased ( p < 0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to their respective NS control groups (NS-2 and NS-3, respectively), with similar trends found in acidic crypt goblet cell count ( Supplementary Table S5 ). Paneth cell count per crypt was significantly increased ( p < 0.05) in NS-3 and Zn groups compared to all other experimental groups, and between NS-3 relative to OE-TaNAS2-3. Paneth cell diameter was significantly increased ( p < 0.05) in NS-1, OE-TaNAS2-1, NS-2, OE-TaNAS2-2, and OE-TaNAS2-3 relative to NS-3, No injection, H₂O injection, and Zn, and between OE-TaNAS-3 relative to the NS-3 group. Effects of OE- TaNAS2A whole wheat flour extracts on the linoleic acid/dihomo-γ-linolenic acid (LA/DGLA) ratio Table 8 Effect of intraamniotic administration of OE- TaNAS2A wheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on chicken erythrocyte LA/DGLA (linoleic acid/dihomo-γ-linolenic acid) on day of hatch . The conversion of LA to DGLA involves potentially Zn-dependent Δ6-desaturase. Values are the means ± SEM. a-e Treatment groups not indicated by the same letter in the same column are significantly different ( p < 0.05) as determined by one-way ANOVA and post-hoc Duncan test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. Treatment Group LA/DGLA (AU) No injection 12.32 ± 1.48 ᵃᵇᶜ H₂O injection 10.25 ± 1.51 ᶜ Zn 14.79 ± 0.13 ᵃᵇᶜ NS-1 14.35 ± 3.09 ᵃᵇᶜ OE-TaNAS2-1 17.92 ± 2.57 ᵃᵇ NS-2 11.13 ± 1.46 ᵇᶜ OE-TaNAS2-2 18.71 ± 2.16 ᵃ NS-3 18.56 ± 3.54 ᵃ OE-TaNAS2-3 19.74 ± 1.14 ᵃ The effects of OE- TaNAS2A whole wheat flour extract on the LA/DGLA ratio, an emerging potential reactive biomarker of Zn physiological status, is depicted in Table 8 . The LA/DGLA ratio was significantly increased in the OE- TaNAS2A groups (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS3-3) relative to the H₂O injection control (Table 8 ). In the OE- TaNAS2A groups relative to their respective NS control groups, there was a trend of increased LA/DGLA ratio. Discussion Biofortification to improve mineral concentration and/or bioavailability in staple crops is a cost-effective and sustainable strategy to complement existing intervention efforts aimed at combating human malnutrition. This study aimed to increase biosynthesis of the metal-chelating molecule nicotianamine (NA) in bread wheat ( Triticum aestivum ), through constitutive overexpression (OE) of an endogenous NA synthase gene ( TaNAS2A ), as a potential Fe and Zn biofortification strategy. Furthermore, this study sought to determine the effects of whole wheat flour derived from field-grown OE- TaNAS2A (NA-biofortified) wheats and their respective null segregant (NS) control wheats on Fe and Zn bioavailability through assessment of biomarkers of Fe and Zn status and gastrointestinal health (duodenal gene expression and histomorphology) in vivo utilizing the embryonic stage of the domestic chicken ( Gallus gallus ). Given the random nature of transgene insertion and the pleiotropic effects of somaclonal variation, this discussion focusses on differences between each independent OE -TaNAS2A transformation event relative to its NS control 56 . Whole wheat flour NA concentration was significantly increased ( p < 0.05) in each OE- TaNAS2A event relative to its respective NS control (Table 3 ). Similarly, DMA concentration was increased in the OE- TaNAS2A wheats relative to their respective NS controls, though this increase was not always statistically significant (Table 3 ). The OE- TaNAS2A whole wheat flours also displayed a trend of increased Zn and Fe concentration relative to their respective NS controls (Table 4 ) but many of these increases were not statistically significant. Overall these findings indicate that constitutive overexpression of TaNAS2A in bread wheat is an effective means of increasing NA concentration in whole wheat flour, however, any increases in DMA, Fe and Zn concentration are not as pronounced nor significant as those observed with constitutive overexpression of the rice OsNAS2 gene 38,40 . Field evaluation of the OE- TaNAS2A wheats demonstrated that key agronomical traits, such as plant height, tiller number, and average spikelet number, did not differ from the respective NS wheats in most cases, suggesting that overexpression of the endogenous TaNAS2A has no adverse impacts on plant phenotype and yield (Table 2 ). Between each OE- TaNAS2A group and its respective NS control group, there were generally no significant differences in the expression of Fe-dependent, Zn-dependent, and inflammatory mediator proteins (Fig. 2 ), potentially due to the short exposure time, in line with our previous intraamniotic administration study assessing extracts of OsNAS2 white wheat flour 12 . The expression of PAT1 (imports NA-bound Fe into enterocytes) was increased in OE-TaNAS-1 relative to NS-1 but decreased in OE-TaNAS-3 relative to NS-3 ( p < 0.05) 39 . In the OE-TaNAS-1 group relative to NS-1, ferroportin (Fe exporter) and IL6 (pro-inflammatory cytokine) expression were increased alongside PAT1. Changes in IL6 expression impact Fe homeostasis and transport, and thus IL6 upregulation could impact PAT1 import of NA-bound Fe and ferroportin export of Fe 57,58 . In the OE-TaNAS-3 group relative to NS-3, both PAT1 and ZIP9 expression were decreased. ZIP9 downregulation is suggestive of increased Zn bioavailability and could be due to OE-TaNAS3 wheat having the largest fold increase in whole wheat flour NA and Zn concentration compared to the other OE- TaNAS2A wheats and their NS controls (Tables 3 and 4 ) 28,59 . Likewise, PAT1 expression downregulation in OE-TaNAS3 relative to NS-3 could be due to increased NA and Zn whole wheat flour concentration as it is possible that PAT1, which transports NA-bound Fe and neutral peptides, could also transport NA-bound Zn as intestinal peptide transporters are hypothesized to import peptide-bound Zn into enterocytes, though further research is required 60 . In the OE-TaNAS-2 group relative to NS-2, hepcidin (hormone which signals for ferroportin degradation) expression was upregulated, which could indicate improvements in Fe status and could be due to OE-TaNAS-2 wheat having the largest fold increase in whole wheat flour Fe concentration compared to the other OE- TaNAS2A wheats and their NS controls 9,61 . Further investigation in long-term studies is required to understand the mechanistic effects of NA/DMA in OE- TaNAS2A wheats on the combination of inflammation and Fe/Zn status. The intraamniotic administration of OE- TaNAS2A whole wheat flour extracts positively altered intestinal functionality, as evidenced by increased enterocyte proliferation (increased villi surface area, crypt depth, and Paneth cell number). Significant increases ( p < 0.05) in villus surface area were found in all OE- TaNAS2A groups (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) relative to their respective NS groups (Table 6 ), in accordance with a previous short-term study utilizing OsNAS2 white wheat flour 12 . Increased villus surface area is indicative of improvements in nutrient digestive and absorptive ability, further highlighting the beneficial effects of NA on intestinal functionality 53,62 . Crypt depth was also increased in each OE- TaNAS2A group relative to its respective NS control group (Table 7 ), suggesting improvements in tissue turnover and cellular proliferation rates 53,62,63 . Further, increased Paneth cell diameter (Table 7 ) was found in each OE- TaNAS2A group relative to its respective NS control group, indicative of improved intestinal regulatory ability due to Paneth cells’ secretory functions in enteric gut microbiota immunoregulation and intestinal stem cell development 64,65 . The combination of increased villi surface area, crypt depth, and Paneth cell size suggest NA-chelated Fe and Zn are highly bioavailable, as indicated by the benefits of OE- TaNAS2A whole wheat flour on intestinal renewal and repair 62,66 . In a previous study with short-term OsNAS2 white flour wheat extract exposure, villi goblet cell number was increased with consumption of OsNAS2 relative to control white wheat flour extract 12 , and a similar trend in increased villi goblet cell number and diameter (Table 6 ) was found with the OE-TaNAS2-2 and OE-TaNAS2-3 groups relative to their respective NS controls (NS-2 and NS-3), suggesting OE- TaNAS2A wheat consumption could increase synthesis of mucin associated with facilitating nutrient hydrolysis and absorption 54,67–69 . However, the opposite trend, decreased villi goblet cell number and diameter, was found in the OE-TaNAS2-1 group relative to NS-1 (Fig. 3 ). IL6 expression was upregulated in the OE-TaNAS2-1 group and IL6 upregulation has previously been demonstrated to impact goblet cell mucin production in vitro 70 . This study explores the potential benefits of OE- TaNAS2A whole wheat flour on improving Fe and Zn bioavailability and intestinal functionality and morphology, providing further evidence that NA is a beneficial phytonutrient in cereal crops. A future long-term study could comprehensively investigate OE- TaNAS2A whole wheat flour composition, as components such as protein, phytate, and fiber could impact mineral bioavailability, though based on previous studies, the concentrations of the aforementioned components are not expected to significantly differ between each OE- TaNAS2A wheat relative to its respective NS control 12,40 . The promising results of this short-term study serve as justification for additional longer-term studies on the beneficial effects of OE- TaNAS2A flour in improving intestinal morphology and functionality. Additionally, future studies focusing on metabolic engineering strategies targeted at utilizing NA and DMA enhancement to increase Fe and Zn bioavailability in staple plant foods are warranted. Future in vivo studies should focus on assessing the benefits associated with NA-chelated Fe and Zn as biofortification strategies to further understand the potential of NA-enhancement in staple crops as a cost-effective and sustainable strategy to combat mineral deficiencies and improved intestinal health in vulnerable populations. Methods Vector construction and generation of wheat transformation events The full-length coding sequence of TaNAS2A ( TraesCS6A02G163100 ) was PCR amplified from wheat ( Triticum aestivum ) cultivar Gladius genomic DNA 47 . Recombination into a modified pMDC32 vector 71 was carried out using the hygromycin phosphotransferase plant-selectable marker gene placed TaNAS2A under transcriptional control of the maize ( Zea mays ) ubiquitin 1 promoter. The T-DNA construct used to transform wheat ( Triticum aestivum ) is shown in ( Supplementary Figure S1 ). Particle bombardment of the construct into immature wheat ( Triticum aestivum ) cultivar Gladius embryos (1.0–1.5 mm in length) was performed at the University of Adelaide using established protocols to generate fifteen independent TaNAS2A transgenic events 47,72 . Following two seasons of growth in the glasshouse (12 h photoperiod, 23°C Day/12°C night, 60% humidity), low-copy transgenic events (T 3 generation) were selected for field analysis based on grain Fe, Zn, NA and DMA concentrations. Confined field trials and whole wheat flour preparation Three OE- TaNAS2A genotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants controls (NS-1, NS-2, and NS-3) were evaluated in confined field trials as previously described 73 at the Dookie Campus of the University of Melbourne (Victoria, Australia) from June to December 2020 (36.3849° S, 145.7070° E). Grain was sown in 1 m 2 plots with three replicate plots per genotype and arranged in a randomized block design (Fig. 1 B and 1 C). Rows were spaced at 30 cm and grains were sown at a rate of 60 kg/ha, with monoammonium phosphate (MAP) applied at sowing (100 kg/Ha) and 200 kg/Ha urea (CH₄N₂O) applied throughout the wheat life cycle. At maturity, average plant height and tiller number were determined from three representative measurements per plot and spike number. Grain yield was calculated from the amount of grain harvested per m 2 plot. Grain subsamples were washed in a 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) solution, rinsed with DI H 2 O, and oven dried for 72 h (70°C) prior to grinding with an IKA Tube Mill control (IKA Works, Inc., Wilmington, NC, USA). Nicotianamine (NA), 2′-deoxymugineic acid (DMA), iron (Fe), and zinc (Zn) concentration determination NA and DMA quantification in OE- TaNAS2A and NS whole wheat flours were performed as previously described 12,74 . Briefly, sequential MeOH (100%) and 18MΩ H₂O samples were derivatized by 9-fluorenylmethoxycarboxyl chloride (FMOC-Cl) and quantified via reverse phase LC-MS on a 1290 Infinity II and 6490 Triple Quadrupole LC/MS system (Agilent Technologies Inc., Santa Clara, CA, USA). For Fe and Zn quantification in whole wheat flour samples, inductively coupled plasma optical emission spectrometry (ICP-OES) was performed on ground samples at the Melbourne Trace Analysis for Chemical, Earth, and Environmental Sciences (TrACEES) Platform at The University of Melbourne (VIC, Australia). Fe and Zn concentration in whole wheat flour water extracts and plasma were determined as previously described 75 . Briefly, the samples were subject to nitric/perchloric acid digestion, followed by inductively coupled plasma-mass spectrometry (ICP-MS) using a Thermo iCAP 6500 series (Thermo Jarrell Ash Corp., Franklin, MA, USA). Preparation of whole wheat flour water extracts and intraamniotic administration solutions Whole wheat flours were dissolved in sterile 18.2 MΩ·cm H₂O (100 g/L), vortexed for 30 s, and incubated in a water bath at 60°C for 60 min. Following incubation, the solutions were cooled at room temperature for 10 min, vortexed for 30 s, and centrifuged at 4000 rpm for 10 mins at 4°C. The resulting supernatant was dialyzed (Spectra/Por ® 7, MWCO 15 kDa, Repligen Corporation, Waltham, Massachusetts, USA) exhaustively against distilled H₂O for 120 h. The dialysate was lyophilized (Millrock Max53 Commercial Freeze Dryer, Millrock, Kingston, NY, USA) to a fine powder and diluted in sterile 18.2 MΩ·cm H₂O (50 g/L), vortexed for 30 s, and incubated in a water bath at 60°C for 60 min. Following incubation, the solutions were cooled at room temperature for 10 min, vortexed for 30s, and centrifuged at 4000 rpm for 10 mins at 4°C. The resulting supernatant formed the 5% whole wheat flour extract for intraamniotic administration. Zinc sulfate solution was prepared by dissolving zinc sulfate in sterile 18.2 MΩ·cm H₂O (20 mg zinc/L). All solutions had an osmolarity value < 320 Osm (see Supplementary Table S2 ), which were measured using a VAPRO Vapor Pressure Osmometer (Wescor, Logan, UT, USA) to prevent chicken embryo dehydration upon injection of the solution. Animals and in vivo (intraamniotic administration) study design Fertile Cornish-cross broiler eggs ( n = 42) were acquired from a commercial hatchery (Moyer’s chicks, Quakertown, PA, USA) and incubated utilizing optimal conditions 76 at the Cornell University Animal Science Poultry Farm hatchery. On embryonic day 17, viable embryos were weighed and divided into nine groups, with all treatment groups randomly assigned eggs of similar weight frequency distribution using a random sequence generator (Random.org, Dublin, Ireland). Injection sites for intraamniotic administration were identified via candling and sterilized with 70% ethanol. 21-gauge needles were utilized to administer 1 mL of each solution per egg into the amniotic fluid for the nine treatment groups: whole wheat flour extract treatment groups (5% extract of OE- TaNAS2A wheats (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregant (NS) control wheats (NS-1, NS-2, and NS-3), two controls (18.2 MΩ·cm DI H₂O injection and no injection), and a Zn control (20 mg/kg zinc sulfate). The Zn control dosage was chosen based on the National Research Council’s (NRC) Nutrient Requirements for Poultry 77 . After injection, the injection holes were sterilized with 70% EtOH and sealed with cellophane tape. The eggs were placed in hatching baskets until hatch, with each treatment equally represented at each incubator location. Immediately upon hatch (day 21), chicks were weighed. Following euthanization by CO 2 exposure, blood was collected. Then, the duodenum, pectoral muscle, and liver were collected, immediately frozen in liquid nitrogen, and then stored at -20°C until analysis. The animal protocol used in this study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Cornell University Institutional Animal Care and Use Committee (IACUC number: 2020-0077, approval date: 20 September 2020). All sections of this report adhere to the ARRIVE Guidelines for reporting animal research 78 . Blood collection Blood was collected via cardiac puncture in microhematocrit heparinized capillary tubes (Fisher Scientific, Waltham, MA, USA) immediately following euthanization. The blood samples were stored on ice until transportation within 4 h to the Tako Laboratory. Whole blood was fractionated by centrifuging at ~ 2000g for 10 minutes at 4°C. Plasma and red blood cell fractions were stored at − 20°C until analysis. Erythrocyte fatty acid extraction Fatty acid (FA) profile was determined after extraction from erythrocytes and saponification. 40 µL of the red blood cell fraction was hemolyzed with 20 µL of 18.2 MΩ·cm H₂O and stored at 4°C overnight. Lipids were extracted using a modified Bligh & Dyer method substituting dichloromethane (DCM) for chloroform 79–81 . Briefly, 190 µL of MeOH was added to 60 µL of the hemolyzed sample, and samples were vortexed for 20 s. Next, 380 µL of DCM was added and vortexed again for 20 s, and 120 µL of DI H₂O was added to induce phase separation. The samples were vortexed for 10 s and allowed to equilibrate at room temperature for 5 min before centrifugation at 8000 g for 10 min. 370 µL of the lower lipid-rich DCM layer was collected, and the solvent was evaporated to dryness under a vacuum. After drying, samples were saponified by adding 350 µL of 0.5 N methanolic NaOH, vortexing for 20 s, and heating in a water bath at 60°C for 10 min. 1 mL hexane was added to extract the free FA, and the samples were vortexed and then centrifuged at 4800 g for 5 min. After centrifugation, the top hexane layer containing free FA was transferred to another tube. The hexane extraction was repeated twice, for a total of 3 mL of free FA-containing hexane. d6-dihomo-γ-linolenic acid ( d6DGLA) was added as internal standard (IS, 50 µg/mL in EtOH) before the samples were evaporated to dryness under vacuum. The samples were resuspended in 1000 µL EtOH prior to being run in the LC-MS. Determination of linoleic acid/dihomo-γ-linolenic acid using LC-MS/MS LA (linoleic acid) and DGLA (dihomo-γ-linolenic acid) standards were prepared as stock solution (100 µg/mL in EtOH). An internal standard, d6DGLA, was also prepared (50 µg/mL in EtOH). LC-MS/MS analysis of LA, DGLA, and d6DGLA was performed using a Luna C18( 2 ) column (3 µm, 100 mm × 2 mm; temp − 40°C; Phenomenex, Torrance, CA, USA) in the Exion LC system (Exion Systems LLC, The Woodlands, TX, USA) coupled with a quadrupole time of flight (QTOF) mass spectrometer (Sciex X500B, Danaher Corporation, Toronto, Canada). The analytes were separated using mobile phase A, (0.1% formic acid in H₂O), and mobile phase B (0.1% formic acid in 100% acetonitrile), with 300 µL/min flow rate and 40°C column temperature. The LC gradient was initiated with 10–35% mobile phase B (MPB) from 0–7 min, 35–95% MPB from 7–7.5 min, 95% MPB from 7.5–11 min, 95–10% MPB from 11–11.5 min, and 10% MPB from 11.5–15 min. The injection volume was 10 µL for the standards and the samples, with 5°C autosampler temperature. The mass spectrometer with an electrospray ionization (ESI) source was operated in negative ion mode. The electrospray voltage was set at 4.5 kV and the heated capillary temperature was set at 350°C. It was operated under the Ion Source gas 1 and 2 at 30 psi, Curtain gas at 20 (AU), and CAD gas at 7 (AU). The declustering potential (DP) was − 80V with 0.25 s accumulation time. The MS full scan measurement was done from m/z 100–1000 in profile mode followed by an MRM HR scan acquired from 0–15 min at collision energy (CE) -40V for each analyte. The ion transitions at m/z 279.23↔TOF fragment mass from m/z 50–350, 305.25↔TOF fragment mass from m/z 50–350 and 311.29↔TOF fragment mass m/z 50–350 were monitored for LA, DGLA, and d6DGLA (IS) respectively in multiple reaction monitor (MRM) mode. CE and DP values were optimized for these transitions. LA and DGLA quantitation were done by integrating peak areas using the MQ4 integration algorithm and MRM transitions (Sciex OS 2.0 software, Danaher Corporation, Toronto, Canada). The peak area ratio for each analyte, LA/IS LA/IS and DGLA/IS, was calculated and then from the two ratios, LA/DGLA was calculated for each sample. Total RNA isolation, cDNA synthesis, primer design, real-time polymerase chain reaction Total RNA was extracted from 30 mg duodenal and hepatic tissue ( n = 3–5) using the Qiagen RNeasy Mini Kit (RNeasy Mini Kit, Qiagen Inc., Valencia, CA, USA) according to manufacturer’s instructions. RNA quantity and quality was measured using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Reverse-transcription to cDNA was done based on manufacturer’s instructions, (Promega-ImProm-II Reverse Transcriptase Kit Catalog #A1250). cDNA concentration and quality were assessed using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). cDNA was stored at -20˚C until use. Primers used in the real-time polymerase chain reactions (RT-qPCR) were designed using Real-Time Primer Design Tool software (IDT DNA, Coralvilla, IA, USA) as was previously described 24,51,52,82 . Primer sequences and accession numbers are shown in Supplementary Table S1 . RT-qPCR reactions were performed using the Bio-RadCFX96 Touch (Hercules, CA, USA), as previously described 24,51,52,82 . All reactions were performed in duplicate under the following optimal conditions: initial denaturation at 95˚C for 30 s, 40 cycles of denaturation at 95˚C for 15 s, various annealing temperatures ( Supplementary Table S1 ) for 30 s and elongation at 60˚C for 30 s. Gene expression levels were obtained from Ct values based on the “second derivative maximum” as computed by the Bio-Rad CFX Maestro Software (Bio-Rad, Hercules, CA, USA) and normalized to 18S expression. Duodenal tissue morphometric analysis Intestinal morphology analysis was performed on duodenal sections as was previously described 52,53,81 . The sections were fixed in fresh 4% ( v / v ) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Sections were cut serially at 5 µm thickness, positioned on glass slides, deparaffinized in xylene, rehydrated in graded alcohol series, and stained with Alcian Blue/Periodic acid-Schiff. Villus height and width, crypt depth, goblet cell diameter, goblet cell type and count within the villus and crypt, Paneth cell number per crypt, and Paneth cell width were measured via light microscopy (EPIX XCAP software standard version, Olympus, Waltham, MA, USA). Per treatment group, three biological samples ( n = 3) and four segments for each biological sample were analyzed. Ten randomly selected villi and crypts were analyzed per segment and cell size measurements and counts were counted in ten randomly selected villi and/or crypts per segment (40 replicates per biological sample). Villus surface area was calculated as previously described 34 . Glycogen concentration analysis as a measurement of energetic status Glycogen content analysis of the pectoral muscle was performed based on an iodine reagent colorimetric method as previously described 27,52,82,83 . Briefly, frozen pectoral muscle was homogenized in 8% perchloric acid and centrifuged at 12,000 x g for 15 min. The supernatant was discarded, and 1 mL of petroleum ether was added. The petroleum ether fraction was discarded and samples from the bottom layer were transferred to a 96-well plate. After 300 µL iodine reagent addition, the samples were incubated at room temperature for 10 min. Samples were read at absorbance 450 nm in a spectrophotometer (Epoch, BioTek, VT, USA) and the glycogen content was calculated according to a standard curve. Statistical analyses Plant phenotype, plant yield, and whole wheat flour nutritional composition results are presented as mean ± standard error of the mean (SEM), n = 3. The Shapiro-Wilk test was utilized to assess distribution normality. To compare between each OE- TaNAS2A wheat and its respective NS wheat, results were analyzed by Student’s t-test, with differences considered statistically significant at p < 0.05. To compare all wheat genotypes, results were analyzed by one-way multiple analysis of variance (ANOVA). A post-hoc Duncan test was used to compare differences between wheat genotypes, with results considered statistically significant at p < 0.05. For the in vivo (intraamniotic administration) study, results are presented as mean ± standard error of the mean (SEM), n = 3–6 biological replicates. Results are presented in tables and heatmaps, and heatmaps were created in Microsoft Excel (version 16.58, Microsoft Corporation, Redmond, WA, USA) based on conditional formatting using color scales. Experimental treatments for the intraamniotic administration experiment were arranged in a completely randomized design. Gene expression was log2 transformed before normality assessment and statistical analyses. The Shapiro-Wilk test was utilized to assess distribution normality, results were analyzed by one-way multiple analysis of variance (ANOVA), and a post-hoc Duncan test was used to compare differences between treatment groups, with results considered statistically significant at p < 0.05. To compare between each OE- TaNAS2A wheat treatment group and its respective NS wheat group, results were analyzed by Student’s t-test, with differences considered statistically significant at p < 0.05. All statistical analyses were performed with R version 4.4.3 software. Declarations Acknowledgements We thank Dr. Ruchika Bhawal and Beth Anderson at the Cornell University BRC Proteomics and Metabolomics Facility (RRID:SCR_021743) for their support in methodology development and analysis of the LA/DGLA fatty acid ratio using LC-MS. We thank Melbourne University’s TrACEES platform for help with ICP-OES analysis of whole wheat flour samples. This project was supported by ARC Linkage project LP190100631. Author contributions statement Plant material engineering, collection, and analysis – J.T.B. and A.A.T.J. In vivo experiment – J.C., C.J., N.K., E.D., and E.T. Acquisition, analysis, and interpretation of data – J.C., N.K., J.T.B., C.J., E.D., A.A.T.J., and E.T. Writing – J.C., J.T.B., A.A.T.J., and E.T. Supervision and funding of plant material engineering, collection, and associated analyses – J.T.B. and A.A.T.J. Supervision and funding of the in vivo experiment and associated analyses – E.T. All authors helped shape the research and reviewed the manuscript. Additional information Competing interests The authors declare no competing interests. Data availability All data is provided within the manuscript or supplementary information files. References WHO. The Global Prevalence of Anaemia in 2011. The World Health Organization (2015). Bailey, R. L., West, K. P., Jr. & Black, R. E. The epidemiology of global micronutrient deficiencies. Ann. Nutr. Metab. 66 Suppl 2 , 22-33 (2015). https://doi.org:10.1159/000371618 WHO/FAO. Vitamin and mineral requirements in human nutrition. (2004). Bongaarts, J. FAO, IFAD, UNICEF, WFP and WHO The State of Food Security and Nutrition in the World 2020. Transforming food systems for affordable healthy diets FAO, 2020, 320 p. Popul. Dev. Rev. 47 , 558-558 (2021). https://doi.org:https://doi.org/10.1111/padr.12418 Wessells, K. R. & Brown, K. H. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4631411","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329529980,"identity":"18395716-3eeb-41a3-b293-98f9a3d9d601","order_by":0,"name":"Elad Tako","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYFAC/o8PPoAZzI0HgAQROtgYjA1ngFmMDURrMZPmIUkLv3xDmrRNjU2+OXsjUEuFdWIDIS2SbQyHrXOOpVnu7DkI1HImnbAWg2OMjbdzGw4bGNxIbDjA2HaYsBb7Y8wM0pYgLfcfArX8I0KLARsbkzQj2Bag94EMwlokjuUwG/YcSzMwOAN0WMKxdGOCWvibzzA++FFjY2Bw/PDBBx9qrGUJakEFCaQpHwWjYBSMglGACwAAq9RBN0XI3ikAAAAASUVORK5CYII=","orcid":"","institution":"Cornell University","correspondingAuthor":true,"prefix":"","firstName":"Elad","middleName":"","lastName":"Tako","suffix":""},{"id":329529981,"identity":"df50eae1-22bc-4748-bad3-823f0af58cd4","order_by":1,"name":"Jacquelyn Cheng","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Jacquelyn","middleName":"","lastName":"Cheng","suffix":""},{"id":329529982,"identity":"f7d24b70-3283-4e0a-af1a-e6f4780c6b36","order_by":2,"name":"Jesse T. 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RB and LB: right and left borders, respectively; UBI-1: maize (Zea Mays) ubiquitin 1 promoter;\u0026nbsp;TaNAS2A: wheat nicotianamine synthase 2A gene (TraesCS6A02G163100); NOS: nopaline synthase terminator; 2 × 35S: dual promoter of the 35S cauliflower mosaic virus gene;\u0026nbsp;hyg: hygromycin phosphotransferase gene; 35S: terminator of the 35S cauliflower mosaic virus gene. (B) Confined field trial of the six field-grown bread wheat genotypes, three OE-TaNAS2A wheats (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregant wheats (NS-1, NS-2, and NS-3) in replicated 1m\u003csup\u003e2\u003c/sup\u003e\u0026nbsp;plots in a randomized block design in Victoria, Australia in 2020. (C) Map and layout of the six wheat genotypes in a randomized block design.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4631411/v1/a10c16f20007786721f3af8b.jpg"},{"id":61011572,"identity":"3201ba74-130a-4f00-b0aa-a20604fcb7e6","added_by":"auto","created_at":"2024-07-24 14:38:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of intraamniotic administration of OE-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaNAS2A\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on chicken duodenal and liver (hepcidin and Δ6-desaturase) Zn- and Fe-related gene expression on day of hatch (Day 21)\u003c/strong\u003e. Gene expression has been normalized to the 18S housekeeping gene, is shown in arbitrary units (AU), and has been log2 transformed. Values are presented as mean ± SEM of 3-6 biological replicates, each with two technical replicates of quantitative RT-PCR. \u003csup\u003ea-c\u003c/sup\u003e Per gene (in the same column), treatments groups not indicated by the same letter are significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) according to one-way ANOVA with post-hoc Duncan test. * Per gene, each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group indicated is significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) relative to its respective null segregant group as determined by Student’s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. DcytB, duodenal cytochrome b; DMT1, divalent metal transporter 1; PAT1, Proton-coupled amino acid transporter 1; ZnT1, zinc transporter 1; ZIP4, zinc transport protein 4; ZIP9, zinc transport protein 9.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4631411/v1/853aa9b07f675e66928686ea.jpg"},{"id":61011571,"identity":"76ef25da-c958-475a-8cb7-4fd54e26f183","added_by":"auto","created_at":"2024-07-24 14:38:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of intraamniotic administration of OE-TaNAS2A (NA-biofortified) and null segregant (control) whole wheat flour extracts on chicken duodenal brush border membrane functionality and inflammatory modulator gene expression on day of hatch (Day 21)\u003c/strong\u003e. Gene expression has been normalized to the 18S housekeeping gene, is shown in arbitrary units (AU), and has been log2 transformed. Values are presented as mean ± SEM of 3-6 biological replicates, each with two technical replicates of quantitative RT-PCR. \u003csup\u003ea-c\u003c/sup\u003e Per gene (in the same column), treatments groups not indicated by the same letter are significantly different (p \u0026lt; 0.05) according to one-way ANOVA with post-hoc Duncan test. * Per gene, each OE-TaNAS2A group indicated is significantly different (p \u0026lt; 0.05) relative to its respective null segregant group as determined by Student’s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄. IL6, interleukin 6; TNF-α, tumor necrosis factor-alpha; NF-\u003csub\u003eΚ\u003c/sub\u003eB1, nuclear factor kappa B subunit 1; SI, sucrase isomaltase; MUC2, mucin 2.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4631411/v1/ab6fdd1fd94f871e2c20a485.jpg"},{"id":71490624,"identity":"046f3726-f894-4cb0-bbe4-8418ecf16781","added_by":"auto","created_at":"2024-12-16 07:24:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1962414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4631411/v1/0f16523f-a1d0-43e7-8b5b-f866cf1c7f74.pdf"},{"id":61011573,"identity":"4837773a-0645-437a-b04a-90c9d9df8b8a","added_by":"auto","created_at":"2024-07-24 14:38:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":409341,"visible":true,"origin":"","legend":"","description":"","filename":"ET062424SupplementaryInformationZnFeWholeWheatSciRepFORSUBMISSION.docx","url":"https://assets-eu.researchsquare.com/files/rs-4631411/v1/dfffe23b1141fea5385453c7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Constitutive overexpression of a nicotianamine synthase gene in bread wheat and in vivo assessment of iron and zinc bioavailability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIron (Fe) deficiency is the most common nutritional disorder in humans, affecting approximately two billion people including 40% of pregnant women and 42% of children below 5 years of age\u003csup\u003e1\u0026ndash;4\u003c/sup\u003e. Zinc (Zn) deficiency is the second most common nutritional disorder in humans, estimated to affect 1\u0026nbsp;billion people, or 17% of the global population\u003csup\u003e2,3,5\u003c/sup\u003e. Iron and Zn deficiencies can have severe consequences on human health, such as depressed immunity, hindered development, and cognitive impairment\u003csup\u003e6\u0026ndash;9\u003c/sup\u003e. Mineral supplementation strategies are efficacious at alleviating severe instances of mineral deficiency, though supplementation efforts in developing regions are often unsuccessful at the population level due to inadequate infrastructure and education\u003csup\u003e10,11\u003c/sup\u003e. Food fortification strategies involve the addition of mineral fortificants to food during processing and are effectively implemented in over 90 countries, however fortification efforts require suitable delivery systems, processing facilities, policies, and constant funding to be effective\u003csup\u003e12,13\u003c/sup\u003e. Further, undesirable sensory characteristics have been associated with Fe fortification of food\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy contrast, biofortification, the process of increasing the nutritional quality of food crops through conventional plant breeding and/or modern biotechnology without sacrificing the preferences of consumers and farmers, represents a more sustainable method for alleviating mineral deficiencies in vulnerable populations\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. Implementing biofortified foods does not require changes to dietary patterns for target populations and can reach rural populations that have poor access to infrastructure\u003csup\u003e19\u003c/sup\u003e. Previous Fe and Zn biofortification efforts in developing regions have demonstrated increases in dietary mineral intakes that are associated with a reduction in human Fe and/or Zn deficiencies\u003csup\u003e18,20\u0026ndash;23\u003c/sup\u003e. The consumption of Fe- and Zn-biofortified foods can increase the colonization of beneficial bacteria in the host gut microbiome and potentially improve intestinal health\u003csup\u003e12,24\u0026ndash;28\u003c/sup\u003e. Crop biofortification with Fe and/or Zn may increase the yield of staple crops under Fe- and/or Zn-deprived soil conditions\u003csup\u003e29\u003c/sup\u003e. Furthermore, economic analyses have demonstrated that biofortification is the most robust and cost-effective strategy for increasing dietary Fe and/or Zn intakes within vulnerable populations in comparison to dietary diversification, supplementation, or food fortification programs\u003csup\u003e10,11,30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCrop-derived bioactive compounds have been shown to improve mineral bioavailability, possess anti-inflammatory properties, and positively modulate the gut microbiome\u003csup\u003e12,31\u0026ndash;34\u003c/sup\u003e. Nicotianamine (NA), a non-protein amino acid-derived plant metabolite, is a naturally occurring chelator of iron (Fe), zinc (Zn), and other transition metals in higher plants and is involved in metal translocation between plant cells, tissues and organs\u003csup\u003e35,36\u003c/sup\u003e. Both NA and its downstream metabolite (2\u0026prime;-deoxymugineic acid, DMA) are major mineral chelators in white wheat flour\u003csup\u003e37\u003c/sup\u003e and are thought to be enhancers of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e Fe bioavailability\u003csup\u003e12,38,39\u003c/sup\u003e. Within intestinal enterocytes (absorptive cells), Fe can be absorbed as an NA-Fe complex via the proton-coupled transporter 1 (PAT1) rather than as inorganic Fe\u003csup\u003e39\u003c/sup\u003e. For these reasons, NA has been described as a bioactive compound that enhances \u003cem\u003ein vivo\u003c/em\u003e Fe bioavailability, and increased biosynthesis of NA/DMA represents a promising strategy for Fe and Zn biofortification of staple crops\u003csup\u003e12,38,40\u0026ndash;42\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) supplies 20% of food calories to the world\u0026rsquo;s population and is the most widely cultivated crop globally\u003csup\u003e43\u003c/sup\u003e. In some low-income communities, wheat accounts for up to 90% of total dietary energy intake\u003csup\u003e44\u003c/sup\u003e, and wheat biofortification efforts aimed at increasing mineral intakes may improve mineral status and reduce the rates of morbidity and mortality in these regions\u003csup\u003e30,45,46\u003c/sup\u003e. We recently assessed \u003cem\u003ein vivo\u003c/em\u003e (\u003cem\u003eGallus gallus\u003c/em\u003e) Fe bioavailability of biofortified white wheat flour, where NA and DMA concentrations were increased via constitutive expression of the rice (\u003cem\u003eOryza sativa\u003c/em\u003e) nicotianamine synthase 2 (\u003cem\u003eOsNAS2\u003c/em\u003e) gene\u003csup\u003e12,40\u003c/sup\u003e. Short-term exposure (intraamniotic administration) to \u003cem\u003eOsNAS2\u003c/em\u003e biofortified white wheat flour extract resulted in improvements in intestinal morphology but not alterations in mineral transport gene expression, and long-term exposure (six-week feeding trial) to biofortified white wheat flour resulted in significant positive changes in host Fe status, brush border membrane functionality and development, and gut microbiota structure and function\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we describe constitutive overexpression (OE) of an endogenous bread wheat nicotianamine synthase 2A (\u003cem\u003eTaNAS2A\u003c/em\u003e) gene as a potential strategy for generating Fe and Zn biofortified wheat and to assess \u003cem\u003ein vivo\u003c/em\u003e Fe and Zn bioavailability within grain produced from field-grown OE-\u003cem\u003eTaNAS2A\u003c/em\u003e and null segregant (NS) control wheat\u003csup\u003e40,47\u003c/sup\u003e. We examine the impact of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e and NS whole wheat flour extracts on mineral (Fe and Zn) status, inflammatory status, and intestinal functionality and morphology, utilizing the \u003cem\u003eG. gallus\u003c/em\u003e model intraamniotic administration approach (embryonic stage, short-term exposure). The \u003cem\u003eG. gallus\u003c/em\u003e system is sensitive to minor modulations in dietary mineral concentration/content and is therefore frequently used to model human dietary mineral bioavailability and absorption\u003csup\u003e24,48\u0026ndash;50\u003c/sup\u003e. The intraamniotic administration method involves injecting extracts into the amniotic fluid at day 17, which are entirely consumed by the embryo prior to hatch, and is widely used to assess the effect of biofortified foods on mineral transport and intestinal functionality and morphology\u003csup\u003e51\u0026ndash;54\u003c/sup\u003e. Furthermore, humans and \u003cem\u003eG. gallus\u003c/em\u003e share\u0026thinsp;\u0026gt;\u0026thinsp;85% gene homology between intestinal brush border membrane proteins involved in mineral transport, further highlighting the relevance of the \u003cem\u003eG. gallus\u003c/em\u003e in assessing mineral bioavailability\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe first objective of this study was to investigate the effect of overexpressing an endogenous \u003cem\u003eTaNAS2A\u003c/em\u003e gene on field-grown bread wheat with respect to phenotype, yield, and grain nutritional composition. The second objective was to assess the effect of intraamniotic administration of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts on biomarkers of Fe and Zn bioavailability utilizing the \u003cem\u003eG. gallus\u003c/em\u003e model.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant production and field evaluation\u003c/h2\u003e \u003cp\u003eBread wheat cultivar (cv.) Gladius transformants (OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats) were generated through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e transformation of a T-DNA construct (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) containing the \u003cem\u003eTaNAS2A\u003c/em\u003e gene under regulatory control of the maize ubiquitin 1 promoter (UBI-1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Three independent OE-\u003cem\u003eTaNAS2A\u003c/em\u003e transformants, pUbi::TaNAS2A 1T, 2T, and 3T (hereafter referred to as OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3, respectively) and their respective null segregants (NS), pUbi::TaNAS2A 1N, 2N, and 3N (hereafter referred to as NS-1, NS-2, and NS-3, respectively, \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e) were evaluated under confined field trial conditions in Victoria, Australia to assess agronomic performance. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows a representative image of replicated 1m\u003csup\u003e2\u003c/sup\u003e plots of the OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat plants and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC displays a map of the randomized block design.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. \u003cb\u003eBread wheat genotypes, description, and reference name within this paper.\u003c/b\u003e \u003cem\u003eTaNAS2A\u003c/em\u003e: wheat nicotianamine synthase 2A gene; NS, null segregant; OE, overexpression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat genotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWheat description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWheat reference name within this paper\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 1N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNull segregant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 1T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverexpression of \u003cem\u003eTaNAS2A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 2N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNull segregant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 2T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverexpression of \u003cem\u003eTaNAS2A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 3N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNull segregant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epUbi::TaNAS2A 3T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverexpression of \u003cem\u003eTaNAS2A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAgromorphological data gathered from field evaluation of the six wheat genotypes is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. No significant differences in plant height or tiller number were detected between genotypes. Average spikelet number was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 relative to NS-2 and plot yield (g per m\u003csup\u003e2\u003c/sup\u003e) was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 and NS-3 relative to NS-1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAgromorphological data of three OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003egenotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, and NS-3)\u003c/b\u003e. \u003csup\u003ea-b\u003c/sup\u003e Per parameter, wheat varieties not indicated by the same letter are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to one-way ANOVA with post-hoc Duncan test. * Per parameter, each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e genotype indicated is significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to its respective null segregant as determined by Student\u0026rsquo;s t-test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat genotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTiller number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage spikelet number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYield (g per m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e106.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.71 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1091.7\u0026thinsp;\u0026plusmn;\u0026thinsp;120.35 ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e123.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1452.1\u0026thinsp;\u0026plusmn;\u0026thinsp;91.21 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e109.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.81 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1256.4\u0026thinsp;\u0026plusmn;\u0026thinsp;55.54 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e120.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.37 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1278.1\u0026thinsp;\u0026plusmn;\u0026thinsp;81.46 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e90.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e110.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1444.6\u0026thinsp;\u0026plusmn;\u0026thinsp;16.06 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e106.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.39 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1272.5\u0026thinsp;\u0026plusmn;\u0026thinsp;75.79 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNicotianamine, DMA, Fe, and Zn concentrations in OE-\u003c/b\u003e \u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewhole wheat flours\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eNicotianamine (NA) and 2\u0026rsquo;-deoxymugineic acid (DMA) concentrations in whole wheat flours of three OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003egenotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, and NS-3)\u003c/b\u003e. \u003csup\u003ea-b\u003c/sup\u003e Per parameter, genotypes not indicated by the same letter are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to one-way ANOVA with post-hoc Duncan test. * Per parameter, each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e genotype indicated is significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to its respective null segregant as determined by Student\u0026rsquo;s t-test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat genotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA (nmol/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e∆NA versus null segregant\u003c/p\u003e \u003cp\u003e(nmol/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDMA (nmol/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e∆DMA versus null segregant (nmol/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 ᵃᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 ᵃᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 ᵃᵇ\u003csup\u003e,\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ᵃᵇ\u003csup\u003e,\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44 ᵇ\u003csup\u003e,\u003c/sup\u003e ᴮ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe concentrations of nicotianamine (NA) and 2\u0026rsquo;-deoxymugineic acid (DMA) in whole wheat flours of the six genotypes are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Whole wheat flour NA concentrations were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to NS-1 and NS-3, and in all OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats relative to their respective NS controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Whole wheat flour DMA concentrations were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to NS-1, and in the OE-TaNAS2-1 and OE-TaNAS2-2 wheats relative to their respective NS controls (NS-1 and NS-2, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eIron (Fe) and zinc (Zn) concentrations in whole wheat flours of three OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003egenotypes (OE-TaNAS2-1, OE-TaNAS2-2, OE-TaNAS2-3) and their respective null segregants (NS-1, NS-2, NS-3).\u003c/b\u003e Values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three technical replicates. \u003csup\u003ea, b\u003c/sup\u003e Per parameter, genotypes not indicated by the same letter are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as determined by one-way ANOVA and post-hoc Duncan test. NS, null segregant; OE, overexpression; NA, nicotianamine; DMA, 2\u0026prime;-deoxymugineic acid.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat genotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFe (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e∆Fe versus null segregant\u003c/p\u003e \u003cp\u003e(mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZn (mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e∆Zn versus null segregant\u003c/p\u003e \u003cp\u003e(mg/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e39.26\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e35.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e46.60\u0026thinsp;\u0026plusmn;\u0026thinsp;11.01 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e34.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e34.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e47.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe concentrations of iron (Fe) and zinc (Zn) in whole wheat flours of the six genotypes are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Whole wheat flour Fe concentrations were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 relative to NS-2 and NS-3. In each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat relative to its respective NS control, there was a trend of increased whole wheat flour Fe concentration. While whole wheat flour Zn concentrations between all wheat genotypes were not significantly different, there was a trend of increased Zn concentration between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat relative to its respective NS control. Fe and Zn concentrations in the whole wheat flour water extracts from all six genotypes are summarized in \u003cb\u003eSupplementary Table S2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of OE-\u003c/b\u003e \u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewhole wheat flour extracts on body weight and pectoral glycogen concentration\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of intraamniotic administration of OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on chick (\u003c/b\u003e\u003cb\u003eGallus gallus\u003c/b\u003e\u003cb\u003e) body weight and pectoral glycogen concentration on day of hatch (Day 21)\u003c/b\u003e. Values are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6. \u003csup\u003ea-c\u003c/sup\u003e Treatment groups not indicated by the same letter in the same column are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) according to one-way ANOVA with post-hoc Duncan test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBodyweight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePectoral Glycogen (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.053\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂O injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.048\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.088\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.147\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e39.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.143\u0026thinsp;\u0026plusmn;\u0026thinsp;0.054 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.076\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e40.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.054\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts on body weight and pectoral glycogen concentration (storage form of glucose) are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Body weight was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 and NS-3 relative to No injection. No significant differences in pectoral glycogen (a measurement of energetic status in poultry) were found between treatment groups. When comparing each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group to its respective NS control, there were no significant differences in body weight or pectoral glycogen. \u003cb\u003eSupplementary Figure S2\u003c/b\u003e depicts the effect OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts on the expression of key gluconeogenesis (metabolic pathway that leads to glucose production) enzymes. No significant differences in PCK1 (phosphoenolpyruvate carboxykinase) expression were found between treatment groups. G6PC1 (glucose-6-phosphatase catalytic subunit 1) expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS-2 and NS-3 compared with OE-TaNAS2-1 and NS-2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRelative gene expression of iron, zinc, BBM (brush border membrane) functionality, and inflammation-related proteins in response to OE-\u003c/b\u003e \u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewhole wheat flour extracts\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the relative expression of Fe metabolism genes in response to OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts. DcytB (duodenal cytochrome B) expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2, NS-2, OE-TaNAS2-3, and NS-3 compared with OE-TaNAS2-1. DMT1 (divalent metal transporter 1) expression was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Zn, NS-2, NS-3, and OE-TaNAS2-3 relative to No Injection, H₂O injection, NS-1, and OE-TaNAS2-1. Ferroportin expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-2, NS-3, and OE-TaNAS2-3 groups relative to No injection, H₂O injection, and NS-1. Hepcidin expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 relative to H₂O injection, Zn, OE-TaNAS2-1, and NS-2. The expression of PAT1 (proton-coupled amino acid transporter 1) was significantly increased in NS-2 and NS-3 relative to No injection, H₂O injection, Zn, and NS-1. Between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group relative to its respective NS control group there were no significant differences in DcytB and DMT1 expression. Ferroportin and PAT1 were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 relative to NS-1, hepcidin was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 relative to NS-2, and PAT1 was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS-3 relative to NS-3.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e also depicts relative expression of Zn-related genes in response to OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts. ZnT1 (Zn transporter 1) expression was significantly increased in NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to NS-1, Zn, No injection, and H₂O injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ZIP4 (Zn transport protein 4) expression was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Zn, NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, OE-TaNAS2-1, No injection, and H₂O injection. ZIP9 (Zn transport protein 9) expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 compared with NS-1, Zn, and H₂O injection. Δ6-desaturase expression was significantly decreased in OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 relative to No injection, H₂O injection, NS-1, and OE-TaNAS2-1. Between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat group relative to its respective NS control group, there were no significant differences in ZnT1 and ZIP4 gene expression. ZIP9 was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 relative to NS-1, Δ6-desaturase was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 relative to NS-2, and ZIP9 was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS-3 relative to NS-3. The effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts on plasma Fe and Zn content is depicted in \u003cb\u003eSupplementary Table S3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicts the relative expression of inflammatory modulator and brush border membrane (BBM) functionality proteins after exposure to OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extract. IL6 (interleukin 6) gene expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, Zn, and H₂O injection. TNF-α (tumor necrosis factor-alpha) expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the NS-2, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 groups relative to the Zn and H₂O injection controls. NF-\u003csub\u003eΚ\u003c/sub\u003eB1 (nuclear factor kappa B subunit 1) expression was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-1 and OE-TaNAS2-1 compared with NS-3 and OE-TaNAS2-3. MUC2 (mucin 2) expression was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-2, NS-3, and OE-TaNAS2-3 relative to NS-1, Zn, and H₂O injection. Between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat group relative to its respective NS control group, there were no significant differences in TNF-α, NF-\u003csub\u003eΚ\u003c/sub\u003eB1, and MUC2 gene expression. IL6 was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1 relative to NS-1 and SI (sucrase-isomaltase) expression was significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 relative to NS-2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of OE-\u003c/b\u003e \u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewhole wheat flour extracts on intestinal morphology: duodenal villi, depth of crypts, goblet cells, and Paneth cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e depicts the effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extract on duodenal villi morphology. Villus surface area was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-1, OE-TaNAS2-2, NS-3, and OE-TaNAS2-3 relative to the No injection, H₂O injection, Zn, NS-1, and NS-2, and between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group relative to its respective NS control group. Villi goblet cell diameter and count were decreased in OE-TaNAS2-1 relative to its NS control group (NS-1). In contrast, villi goblet cell diameter and count were increased in OE-TaNAS2-2 and OE-TaNAS2-3 relative to their NS control groups (NS-2 and NS-3, respectively). Acidic goblet cells were the predominant goblet cell type within the villi and the trends in acidic villi goblet cell count were consistent with the total villi goblet cell count (\u003cb\u003eSupplementary Table S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of intraamniotic administration of OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003e(NA-biofortified) and null segregant (control) whole wheat flour extracts on duodenal villi and villi goblet cells\u003c/b\u003e. Values are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Three biological samples per treatment group and four segments for each biological sample were analyzed. Ten randomly selected villi were analyzed per segment and cell size measurements and counts were counted in ten randomly selected villi per segment (40 replicates per biological sample). \u003csup\u003ea-e\u003c/sup\u003e Treatment groups not indicated by the same letter in the same column are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as determined by one-way ANOVA and post-hoc Duncan test. * Per parameter, each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group indicated is significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to its respective null segregant group as determined by Student\u0026rsquo;s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus Surface Area (\u0026micro;m\u0026sup2;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVilli Goblet Diameter (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVilli Goblet Cell Count (unit)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e138.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 ᵉ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂O injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e130.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24 ᵈᵉ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e121.43\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64 ᵉ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e144.88\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e182.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.62 ᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e27.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 ᵈ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e136.93\u0026thinsp;\u0026plusmn;\u0026thinsp;6.24 ᵈᵉ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e28.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 ᶜᵈ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e197.76\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᶜ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e163.60\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e205.34\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 ᶜ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e34.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 ᵃᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of intraamniotic administration of OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on duodenal crypts, crypt goblet cells, and Paneth cells\u003c/b\u003e. Values are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Three biological samples and four segments for each biological sample were analyzed per treatment group. Ten randomly selected crypts were analyzed per segment and cell size measurements and counts were counted in ten randomly selected crypts per segment (40 replicates per biological sample). \u003csup\u003ea-e\u003c/sup\u003e Treatment groups not indicated by the same letter in the same column are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as determined by one-way ANOVA and post-hoc Duncan test. * Per parameter, each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group indicated is significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to its respective null segregant group as determined by Student\u0026rsquo;s t-test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrypt Depth (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrypt Goblet Diameter (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCrypt Goblet Cell Count (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePaneth Cell Count per Crypt (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePaneth Cell Diameter (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵈ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂O injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 ᶜᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵈ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵉ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93 ᶜᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 ᶜᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 ᶜᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᵈ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 ᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 ᵇᶜ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 ᵈᵉ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 ᵈ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ᵇ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ᵃ\u003csup\u003e,\u003c/sup\u003e *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e7\u003c/span\u003e depicts the effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extract on duodenal crypts, crypt goblet cells, and Paneth cells. Crypt depth was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-3 and OE-TaNAS2-3 relative to NS-1 and OE-TaNAS2-1, and between OE-TaNAS2-2 and OE-TaNAS2-3 relative to their respective NS control groups (NS-2 and NS-3, respectively). Crypt goblet diameter was significantly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in all OE-\u003cem\u003eTaNAS2A\u003c/em\u003e groups relative to No injection and H₂O injection controls, and between OE-TaNAS2-1 and OE-TaNAS2-3 relative to their respective NS control groups (NS-1 and NS-3, respectively). Crypt goblet cell count was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in OE-TaNAS2-2 and OE-TaNAS2-3 relative to their respective NS control groups (NS-2 and NS-3, respectively), with similar trends found in acidic crypt goblet cell count (\u003cb\u003eSupplementary Table S5\u003c/b\u003e). Paneth cell count per crypt was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-3 and Zn groups compared to all other experimental groups, and between NS-3 relative to OE-TaNAS2-3. Paneth cell diameter was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in NS-1, OE-TaNAS2-1, NS-2, OE-TaNAS2-2, and OE-TaNAS2-3 relative to NS-3, No injection, H₂O injection, and Zn, and between OE-TaNAS-3 relative to the NS-3 group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of OE-\u003c/b\u003e \u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewhole wheat flour extracts on the linoleic acid/dihomo-γ-linolenic acid (LA/DGLA) ratio\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of intraamniotic administration of OE-\u003c/b\u003e\u003cb\u003eTaNAS2A\u003c/b\u003e \u003cb\u003ewheat (NA-biofortified) and null segregant (control) whole wheat flour extracts on chicken erythrocyte LA/DGLA (linoleic acid/dihomo-γ-linolenic acid) on day of hatch\u003c/b\u003e. The conversion of LA to DGLA involves potentially Zn-dependent Δ6-desaturase. Values are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. \u003csup\u003ea-e\u003c/sup\u003e Treatment groups not indicated by the same letter in the same column are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) as determined by one-way ANOVA and post-hoc Duncan test. NS, null segregant; OE, overexpression; Zn, 20 mg/kg ZnSO₄.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLA/DGLA (AU)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂O injection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51 ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09 ᵃᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57 ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46 ᵇᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNS-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOE-TaNAS2-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extract on the LA/DGLA ratio, an emerging potential reactive biomarker of Zn physiological status, is depicted in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The LA/DGLA ratio was significantly increased in the OE-\u003cem\u003eTaNAS2A\u003c/em\u003e groups (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS3-3) relative to the H₂O injection control (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In the OE-\u003cem\u003eTaNAS2A\u003c/em\u003e groups relative to their respective NS control groups, there was a trend of increased LA/DGLA ratio.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBiofortification to improve mineral concentration and/or bioavailability in staple crops is a cost-effective and sustainable strategy to complement existing intervention efforts aimed at combating human malnutrition. This study aimed to increase biosynthesis of the metal-chelating molecule nicotianamine (NA) in bread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e), through constitutive overexpression (OE) of an endogenous NA synthase gene (\u003cem\u003eTaNAS2A\u003c/em\u003e), as a potential Fe and Zn biofortification strategy. Furthermore, this study sought to determine the effects of whole wheat flour derived from field-grown OE-\u003cem\u003eTaNAS2A\u003c/em\u003e (NA-biofortified) wheats and their respective null segregant (NS) control wheats on Fe and Zn bioavailability through assessment of biomarkers of Fe and Zn status and gastrointestinal health (duodenal gene expression and histomorphology) \u003cem\u003ein vivo\u003c/em\u003e utilizing the embryonic stage of the domestic chicken (\u003cem\u003eGallus gallus\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eGiven the random nature of transgene insertion and the pleiotropic effects of somaclonal variation, this discussion focusses on differences between each independent OE\u003cem\u003e-TaNAS2A\u003c/em\u003e transformation event relative to its NS control\u003csup\u003e56\u003c/sup\u003e. Whole wheat flour NA concentration was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e event relative to its respective NS control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, DMA concentration was increased in the OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats relative to their respective NS controls, though this increase was not always statistically significant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flours also displayed a trend of increased Zn and Fe concentration relative to their respective NS controls (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) but many of these increases were not statistically significant. Overall these findings indicate that constitutive overexpression of \u003cem\u003eTaNAS2A\u003c/em\u003e in bread wheat is an effective means of increasing NA concentration in whole wheat flour, however, any increases in DMA, Fe and Zn concentration are not as pronounced nor significant as those observed with constitutive overexpression of the rice \u003cem\u003eOsNAS2\u003c/em\u003e gene\u003csup\u003e38,40\u003c/sup\u003e. Field evaluation of the OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats demonstrated that key agronomical traits, such as plant height, tiller number, and average spikelet number, did not differ from the respective NS wheats in most cases, suggesting that overexpression of the endogenous \u003cem\u003eTaNAS2A\u003c/em\u003e has no adverse impacts on plant phenotype and yield (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBetween each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group and its respective NS control group, there were generally no significant differences in the expression of Fe-dependent, Zn-dependent, and inflammatory mediator proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), potentially due to the short exposure time, in line with our previous intraamniotic administration study assessing extracts of \u003cem\u003eOsNAS2\u003c/em\u003e white wheat flour\u003csup\u003e12\u003c/sup\u003e. The expression of PAT1 (imports NA-bound Fe into enterocytes) was increased in OE-TaNAS-1 relative to NS-1 but decreased in OE-TaNAS-3 relative to NS-3 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003csup\u003e39\u003c/sup\u003e. In the OE-TaNAS-1 group relative to NS-1, ferroportin (Fe exporter) and IL6 (pro-inflammatory cytokine) expression were increased alongside PAT1. Changes in IL6 expression impact Fe homeostasis and transport, and thus IL6 upregulation could impact PAT1 import of NA-bound Fe and ferroportin export of Fe\u003csup\u003e57,58\u003c/sup\u003e. In the OE-TaNAS-3 group relative to NS-3, both PAT1 and ZIP9 expression were decreased. ZIP9 downregulation is suggestive of increased Zn bioavailability and could be due to OE-TaNAS3 wheat having the largest fold increase in whole wheat flour NA and Zn concentration compared to the other OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats and their NS controls (Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003csup\u003e28,59\u003c/sup\u003e. Likewise, PAT1 expression downregulation in OE-TaNAS3 relative to NS-3 could be due to increased NA and Zn whole wheat flour concentration as it is possible that PAT1, which transports NA-bound Fe and neutral peptides, could also transport NA-bound Zn as intestinal peptide transporters are hypothesized to import peptide-bound Zn into enterocytes, though further research is required\u003csup\u003e60\u003c/sup\u003e. In the OE-TaNAS-2 group relative to NS-2, hepcidin (hormone which signals for ferroportin degradation) expression was upregulated, which could indicate improvements in Fe status and could be due to OE-TaNAS-2 wheat having the largest fold increase in whole wheat flour Fe concentration compared to the other OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats and their NS controls\u003csup\u003e9,61\u003c/sup\u003e. Further investigation in long-term studies is required to understand the mechanistic effects of NA/DMA in OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats on the combination of inflammation and Fe/Zn status.\u003c/p\u003e \u003cp\u003eThe intraamniotic administration of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour extracts positively altered intestinal functionality, as evidenced by increased enterocyte proliferation (increased villi surface area, crypt depth, and Paneth cell number). Significant increases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in villus surface area were found in all OE-\u003cem\u003eTaNAS2A\u003c/em\u003e groups (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) relative to their respective NS groups (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e), in accordance with a previous short-term study utilizing \u003cem\u003eOsNAS2\u003c/em\u003e white wheat flour\u003csup\u003e12\u003c/sup\u003e. Increased villus surface area is indicative of improvements in nutrient digestive and absorptive ability, further highlighting the beneficial effects of NA on intestinal functionality\u003csup\u003e53,62\u003c/sup\u003e. Crypt depth was also increased in each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group relative to its respective NS control group (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting improvements in tissue turnover and cellular proliferation rates\u003csup\u003e53,62,63\u003c/sup\u003e. Further, increased Paneth cell diameter (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e7\u003c/span\u003e) was found in each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e group relative to its respective NS control group, indicative of improved intestinal regulatory ability due to Paneth cells\u0026rsquo; secretory functions in enteric gut microbiota immunoregulation and intestinal stem cell development\u003csup\u003e64,65\u003c/sup\u003e. The combination of increased villi surface area, crypt depth, and Paneth cell size suggest NA-chelated Fe and Zn are highly bioavailable, as indicated by the benefits of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour on intestinal renewal and repair\u003csup\u003e62,66\u003c/sup\u003e. In a previous study with short-term \u003cem\u003eOsNAS2\u003c/em\u003e white flour wheat extract exposure, villi goblet cell number was increased with consumption of \u003cem\u003eOsNAS2\u003c/em\u003e relative to control white wheat flour extract\u003csup\u003e12\u003c/sup\u003e, and a similar trend in increased villi goblet cell number and diameter (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e) was found with the OE-TaNAS2-2 and OE-TaNAS2-3 groups relative to their respective NS controls (NS-2 and NS-3), suggesting OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat consumption could increase synthesis of mucin associated with facilitating nutrient hydrolysis and absorption\u003csup\u003e54,67\u0026ndash;69\u003c/sup\u003e. However, the opposite trend, decreased villi goblet cell number and diameter, was found in the OE-TaNAS2-1 group relative to NS-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). IL6 expression was upregulated in the OE-TaNAS2-1 group and IL6 upregulation has previously been demonstrated to impact goblet cell mucin production \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e70\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study explores the potential benefits of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour on improving Fe and Zn bioavailability and intestinal functionality and morphology, providing further evidence that NA is a beneficial phytonutrient in cereal crops. A future long-term study could comprehensively investigate OE-\u003cem\u003eTaNAS2A\u003c/em\u003e whole wheat flour composition, as components such as protein, phytate, and fiber could impact mineral bioavailability, though based on previous studies, the concentrations of the aforementioned components are not expected to significantly differ between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat relative to its respective NS control\u003csup\u003e12,40\u003c/sup\u003e. The promising results of this short-term study serve as justification for additional longer-term studies on the beneficial effects of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e flour in improving intestinal morphology and functionality. Additionally, future studies focusing on metabolic engineering strategies targeted at utilizing NA and DMA enhancement to increase Fe and Zn bioavailability in staple plant foods are warranted. Future \u003cem\u003ein vivo\u003c/em\u003e studies should focus on assessing the benefits associated with NA-chelated Fe and Zn as biofortification strategies to further understand the potential of NA-enhancement in staple crops as a cost-effective and sustainable strategy to combat mineral deficiencies and improved intestinal health in vulnerable populations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVector construction and generation of wheat transformation events\u003c/h2\u003e \u003cp\u003eThe full-length coding sequence of \u003cem\u003eTaNAS2A\u003c/em\u003e (\u003cem\u003eTraesCS6A02G163100\u003c/em\u003e) was PCR amplified from wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) cultivar Gladius genomic DNA\u003csup\u003e47\u003c/sup\u003e. Recombination into a modified pMDC32 vector\u003csup\u003e71\u003c/sup\u003e was carried out using the hygromycin phosphotransferase plant-selectable marker gene placed \u003cem\u003eTaNAS2A\u003c/em\u003e under transcriptional control of the maize (\u003cem\u003eZea mays\u003c/em\u003e) ubiquitin 1 promoter. The T-DNA construct used to transform wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) is shown in (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Particle bombardment of the construct into immature wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) cultivar Gladius embryos (1.0\u0026ndash;1.5 mm in length) was performed at the University of Adelaide using established protocols to generate fifteen independent \u003cem\u003eTaNAS2A\u003c/em\u003e transgenic events\u003csup\u003e47,72\u003c/sup\u003e. Following two seasons of growth in the glasshouse (12 h photoperiod, 23\u0026deg;C Day/12\u0026deg;C night, 60% humidity), low-copy transgenic events (T\u003csub\u003e3\u003c/sub\u003e generation) were selected for field analysis based on grain Fe, Zn, NA and DMA concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eConfined field trials and whole wheat flour preparation\u003c/h2\u003e \u003cp\u003eThree OE-\u003cem\u003eTaNAS2A\u003c/em\u003e genotypes (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregants controls (NS-1, NS-2, and NS-3) were evaluated in confined field trials as previously described\u003csup\u003e73\u003c/sup\u003e at the Dookie Campus of the University of Melbourne (Victoria, Australia) from June to December 2020 (36.3849\u0026deg; S, 145.7070\u0026deg; E). Grain was sown in 1 m\u003csup\u003e2\u003c/sup\u003e plots with three replicate plots per genotype and arranged in a randomized block design (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Rows were spaced at 30 cm and grains were sown at a rate of 60 kg/ha, with monoammonium phosphate (MAP) applied at sowing (100 kg/Ha) and 200 kg/Ha urea (CH₄N₂O) applied throughout the wheat life cycle. At maturity, average plant height and tiller number were determined from three representative measurements per plot and spike number. Grain yield was calculated from the amount of grain harvested per m\u003csup\u003e2\u003c/sup\u003e plot. Grain subsamples were washed in a 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) solution, rinsed with DI H\u003csub\u003e2\u003c/sub\u003eO, and oven dried for 72 h (70\u0026deg;C) prior to grinding with an IKA Tube Mill control (IKA Works, Inc., Wilmington, NC, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNicotianamine (NA), 2\u0026prime;-deoxymugineic acid (DMA), iron (Fe), and zinc (Zn) concentration determination\u003c/h2\u003e \u003cp\u003eNA and DMA quantification in OE-\u003cem\u003eTaNAS2A\u003c/em\u003e and NS whole wheat flours were performed as previously described\u003csup\u003e12,74\u003c/sup\u003e. Briefly, sequential MeOH (100%) and 18MΩ H₂O samples were derivatized by 9-fluorenylmethoxycarboxyl chloride (FMOC-Cl) and quantified via reverse phase LC-MS on a 1290 Infinity II and 6490 Triple Quadrupole LC/MS system (Agilent Technologies Inc., Santa Clara, CA, USA). For Fe and Zn quantification in whole wheat flour samples, inductively coupled plasma optical emission spectrometry (ICP-OES) was performed on ground samples at the Melbourne Trace Analysis for Chemical, Earth, and Environmental Sciences (TrACEES) Platform at The University of Melbourne (VIC, Australia). Fe and Zn concentration in whole wheat flour water extracts and plasma were determined as previously described\u003csup\u003e75\u003c/sup\u003e. Briefly, the samples were subject to nitric/perchloric acid digestion, followed by inductively coupled plasma-mass spectrometry (ICP-MS) using a Thermo iCAP 6500 series (Thermo Jarrell Ash Corp., Franklin, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of whole wheat flour water extracts and intraamniotic administration solutions\u003c/h2\u003e \u003cp\u003eWhole wheat flours were dissolved in sterile 18.2 MΩ\u0026middot;cm H₂O (100 g/L), vortexed for 30 s, and incubated in a water bath at 60\u0026deg;C for 60 min. Following incubation, the solutions were cooled at room temperature for 10 min, vortexed for 30 s, and centrifuged at 4000 rpm for 10 mins at 4\u0026deg;C. The resulting supernatant was dialyzed (Spectra/Por\u003csup\u003e\u0026reg;\u003c/sup\u003e 7, MWCO 15 kDa, Repligen Corporation, Waltham, Massachusetts, USA) exhaustively against distilled H₂O for 120 h. The dialysate was lyophilized (Millrock Max53 Commercial Freeze Dryer, Millrock, Kingston, NY, USA) to a fine powder and diluted in sterile 18.2 MΩ\u0026middot;cm H₂O (50 g/L), vortexed for 30 s, and incubated in a water bath at 60\u0026deg;C for 60 min. Following incubation, the solutions were cooled at room temperature for 10 min, vortexed for 30s, and centrifuged at 4000 rpm for 10 mins at 4\u0026deg;C. The resulting supernatant formed the 5% whole wheat flour extract for intraamniotic administration. Zinc sulfate solution was prepared by dissolving zinc sulfate in sterile 18.2 MΩ\u0026middot;cm H₂O (20 mg zinc/L). All solutions had an osmolarity value\u0026thinsp;\u0026lt;\u0026thinsp;320 Osm (see \u003cb\u003eSupplementary Table S2\u003c/b\u003e), which were measured using a VAPRO Vapor Pressure Osmometer (Wescor, Logan, UT, USA) to prevent chicken embryo dehydration upon injection of the solution.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimals and\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003e(intraamniotic administration) study design\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFertile Cornish-cross broiler eggs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;42) were acquired from a commercial hatchery (Moyer\u0026rsquo;s chicks, Quakertown, PA, USA) and incubated utilizing optimal conditions\u003csup\u003e76\u003c/sup\u003e at the Cornell University Animal Science Poultry Farm hatchery. On embryonic day 17, viable embryos were weighed and divided into nine groups, with all treatment groups randomly assigned eggs of similar weight frequency distribution using a random sequence generator (Random.org, Dublin, Ireland). Injection sites for intraamniotic administration were identified via candling and sterilized with 70% ethanol. 21-gauge needles were utilized to administer 1 mL of each solution per egg into the amniotic fluid for the nine treatment groups: whole wheat flour extract treatment groups (5% extract of OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheats (OE-TaNAS2-1, OE-TaNAS2-2, and OE-TaNAS2-3) and their respective null segregant (NS) control wheats (NS-1, NS-2, and NS-3), two controls (18.2 MΩ\u0026middot;cm DI H₂O injection and no injection), and a Zn control (20 mg/kg zinc sulfate). The Zn control dosage was chosen based on the National Research Council\u0026rsquo;s (NRC) Nutrient Requirements for Poultry\u003csup\u003e77\u003c/sup\u003e. After injection, the injection holes were sterilized with 70% EtOH and sealed with cellophane tape. The eggs were placed in hatching baskets until hatch, with each treatment equally represented at each incubator location. Immediately upon hatch (day 21), chicks were weighed. Following euthanization by CO\u003csub\u003e2\u003c/sub\u003e exposure, blood was collected. Then, the duodenum, pectoral muscle, and liver were collected, immediately frozen in liquid nitrogen, and then stored at -20\u0026deg;C until analysis. The animal protocol used in this study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Cornell University Institutional Animal Care and Use Committee (IACUC number: 2020-0077, approval date: 20 September 2020). All sections of this report adhere to the ARRIVE Guidelines for reporting animal research\u003csup\u003e78\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eBlood collection\u003c/h2\u003e \u003cp\u003eBlood was collected via cardiac puncture in microhematocrit heparinized capillary tubes (Fisher Scientific, Waltham, MA, USA) immediately following euthanization. The blood samples were stored on ice until transportation within 4 h to the Tako Laboratory. Whole blood was fractionated by centrifuging at ~\u0026thinsp;2000g for 10 minutes at 4\u0026deg;C. Plasma and red blood cell fractions were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eErythrocyte fatty acid extraction\u003c/h2\u003e \u003cp\u003eFatty acid (FA) profile was determined after extraction from erythrocytes and saponification. 40 \u0026micro;L of the red blood cell fraction was hemolyzed with 20 \u0026micro;L of 18.2 MΩ\u0026middot;cm H₂O and stored at 4\u0026deg;C overnight. Lipids were extracted using a modified Bligh \u0026amp; Dyer method substituting dichloromethane (DCM) for chloroform\u003csup\u003e79\u0026ndash;81\u003c/sup\u003e. Briefly, 190 \u0026micro;L of MeOH was added to 60 \u0026micro;L of the hemolyzed sample, and samples were vortexed for 20 s. Next, 380 \u0026micro;L of DCM was added and vortexed again for 20 s, and 120 \u0026micro;L of DI H₂O was added to induce phase separation. The samples were vortexed for 10 s and allowed to equilibrate at room temperature for 5 min before centrifugation at 8000 g for 10 min. 370 \u0026micro;L of the lower lipid-rich DCM layer was collected, and the solvent was evaporated to dryness under a vacuum. After drying, samples were saponified by adding 350 \u0026micro;L of 0.5 N methanolic NaOH, vortexing for 20 s, and heating in a water bath at 60\u0026deg;C for 10 min. 1 mL hexane was added to extract the free FA, and the samples were vortexed and then centrifuged at 4800 g for 5 min. After centrifugation, the top hexane layer containing free FA was transferred to another tube. The hexane extraction was repeated twice, for a total of 3 mL of free FA-containing hexane. d6-dihomo-γ-linolenic acid \u003cb\u003e(\u003c/b\u003ed6DGLA) was added as internal standard (IS, 50 \u0026micro;g/mL in EtOH) before the samples were evaporated to dryness under vacuum. The samples were resuspended in 1000 \u0026micro;L EtOH prior to being run in the LC-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of linoleic acid/dihomo-γ-linolenic acid using LC-MS/MS\u003c/h2\u003e \u003cp\u003eLA (linoleic acid) and DGLA (dihomo-γ-linolenic acid) standards were prepared as stock solution (100 \u0026micro;g/mL in EtOH). An internal standard, d6DGLA, was also prepared (50 \u0026micro;g/mL in EtOH). LC-MS/MS analysis of LA, DGLA, and d6DGLA was performed using a Luna C18(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) column (3 \u0026micro;m, 100 mm \u0026times; 2 mm; temp \u0026minus;\u0026thinsp;40\u0026deg;C; Phenomenex, Torrance, CA, USA) in the Exion LC system (Exion Systems LLC, The Woodlands, TX, USA) coupled with a quadrupole time of flight (QTOF) mass spectrometer (Sciex X500B, Danaher Corporation, Toronto, Canada). The analytes were separated using mobile phase A, (0.1% formic acid in H₂O), and mobile phase B (0.1% formic acid in 100% acetonitrile), with 300 \u0026micro;L/min flow rate and 40\u0026deg;C column temperature. The LC gradient was initiated with 10\u0026ndash;35% mobile phase B (MPB) from 0\u0026ndash;7 min, 35\u0026ndash;95% MPB from 7\u0026ndash;7.5 min, 95% MPB from 7.5\u0026ndash;11 min, 95\u0026ndash;10% MPB from 11\u0026ndash;11.5 min, and 10% MPB from 11.5\u0026ndash;15 min. The injection volume was 10 \u0026micro;L for the standards and the samples, with 5\u0026deg;C autosampler temperature. The mass spectrometer with an electrospray ionization (ESI) source was operated in negative ion mode. The electrospray voltage was set at 4.5 kV and the heated capillary temperature was set at 350\u0026deg;C. It was operated under the Ion Source gas 1 and 2 at 30 psi, Curtain gas at 20 (AU), and CAD gas at 7 (AU). The declustering potential (DP) was \u0026minus;\u0026thinsp;80V with 0.25 s accumulation time. The MS full scan measurement was done from m/z 100\u0026ndash;1000 in profile mode followed by an MRM HR scan acquired from 0\u0026ndash;15 min at collision energy (CE) -40V for each analyte. The ion transitions at m/z 279.23\u0026harr;TOF fragment mass from m/z 50\u0026ndash;350, 305.25\u0026harr;TOF fragment mass from m/z 50\u0026ndash;350 and 311.29\u0026harr;TOF fragment mass m/z 50\u0026ndash;350 were monitored for LA, DGLA, and d6DGLA (IS) respectively in multiple reaction monitor (MRM) mode. CE and DP values were optimized for these transitions. LA and DGLA quantitation were done by integrating peak areas using the MQ4 integration algorithm and MRM transitions (Sciex OS 2.0 software, Danaher Corporation, Toronto, Canada). The peak area ratio for each analyte, LA/IS LA/IS and DGLA/IS, was calculated and then from the two ratios, LA/DGLA was calculated for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation, cDNA synthesis, primer design, real-time polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 30 mg duodenal and hepatic tissue (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u0026ndash;5) using the Qiagen RNeasy Mini Kit (RNeasy Mini Kit, Qiagen Inc., Valencia, CA, USA) according to manufacturer\u0026rsquo;s instructions. RNA quantity and quality was measured using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Reverse-transcription to cDNA was done based on manufacturer\u0026rsquo;s instructions, (Promega-ImProm-II Reverse Transcriptase Kit Catalog #A1250). cDNA concentration and quality were assessed using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). cDNA was stored at -20˚C until use. Primers used in the real-time polymerase chain reactions (RT-qPCR) were designed using Real-Time Primer Design Tool software (IDT DNA, Coralvilla, IA, USA) as was previously described\u003csup\u003e24,51,52,82\u003c/sup\u003e. Primer sequences and accession numbers are shown in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. RT-qPCR reactions were performed using the Bio-RadCFX96 Touch (Hercules, CA, USA), as previously described\u003csup\u003e24,51,52,82\u003c/sup\u003e. All reactions were performed in duplicate under the following optimal conditions: initial denaturation at 95˚C for 30 s, 40 cycles of denaturation at 95˚C for 15 s, various annealing temperatures (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) for 30 s and elongation at 60˚C for 30 s. Gene expression levels were obtained from Ct values based on the \u0026ldquo;second derivative maximum\u0026rdquo; as computed by the Bio-Rad CFX Maestro Software (Bio-Rad, Hercules, CA, USA) and normalized to 18S expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDuodenal tissue morphometric analysis\u003c/h2\u003e \u003cp\u003eIntestinal morphology analysis was performed on duodenal sections as was previously described\u003csup\u003e52,53,81\u003c/sup\u003e. The sections were fixed in fresh 4% (\u003cem\u003ev\u003c/em\u003e/\u003cem\u003ev\u003c/em\u003e) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Sections were cut serially at 5 \u0026micro;m thickness, positioned on glass slides, deparaffinized in xylene, rehydrated in graded alcohol series, and stained with Alcian Blue/Periodic acid-Schiff. Villus height and width, crypt depth, goblet cell diameter, goblet cell type and count within the villus and crypt, Paneth cell number per crypt, and Paneth cell width were measured via light microscopy (EPIX XCAP software standard version, Olympus, Waltham, MA, USA). Per treatment group, three biological samples (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) and four segments for each biological sample were analyzed. Ten randomly selected villi and crypts were analyzed per segment and cell size measurements and counts were counted in ten randomly selected villi and/or crypts per segment (40 replicates per biological sample). Villus surface area was calculated as previously described\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGlycogen concentration analysis as a measurement of energetic status\u003c/h2\u003e \u003cp\u003eGlycogen content analysis of the pectoral muscle was performed based on an iodine reagent colorimetric method as previously described\u003csup\u003e27,52,82,83\u003c/sup\u003e. Briefly, frozen pectoral muscle was homogenized in 8% perchloric acid and centrifuged at 12,000 x \u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant was discarded, and 1 mL of petroleum ether was added. The petroleum ether fraction was discarded and samples from the bottom layer were transferred to a 96-well plate. After 300 \u0026micro;L iodine reagent addition, the samples were incubated at room temperature for 10 min. Samples were read at absorbance 450 nm in a spectrophotometer (Epoch, BioTek, VT, USA) and the glycogen content was calculated according to a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003ePlant phenotype, plant yield, and whole wheat flour nutritional composition results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3. The Shapiro-Wilk test was utilized to assess distribution normality. To compare between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat and its respective NS wheat, results were analyzed by Student\u0026rsquo;s t-test, with differences considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To compare all wheat genotypes, results were analyzed by one-way multiple analysis of variance (ANOVA). A post-hoc Duncan test was used to compare differences between wheat genotypes, with results considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For the \u003cem\u003ein vivo\u003c/em\u003e (intraamniotic administration) study, results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3\u0026ndash;6 biological replicates. Results are presented in tables and heatmaps, and heatmaps were created in Microsoft Excel (version 16.58, Microsoft Corporation, Redmond, WA, USA) based on conditional formatting using color scales. Experimental treatments for the intraamniotic administration experiment were arranged in a completely randomized design. Gene expression was log2 transformed before normality assessment and statistical analyses. The Shapiro-Wilk test was utilized to assess distribution normality, results were analyzed by one-way multiple analysis of variance (ANOVA), and a post-hoc Duncan test was used to compare differences between treatment groups, with results considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. To compare between each OE-\u003cem\u003eTaNAS2A\u003c/em\u003e wheat treatment group and its respective NS wheat group, results were analyzed by Student\u0026rsquo;s t-test, with differences considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed with R version 4.4.3 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Ruchika Bhawal and Beth Anderson at the Cornell University BRC Proteomics and Metabolomics Facility (RRID:SCR_021743) for their support in methodology development and analysis of the LA/DGLA fatty acid ratio using LC-MS. We thank Melbourne University’s TrACEES platform for help with ICP-OES analysis of whole wheat flour samples. This project was supported by ARC Linkage project LP190100631.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant material engineering, collection, and analysis – J.T.B. and A.A.T.J. In vivo experiment – J.C., C.J., N.K., E.D., and E.T. Acquisition, analysis, and interpretation of data – J.C., N.K., J.T.B., C.J., E.D., A.A.T.J., and E.T. Writing – J.C., J.T.B., A.A.T.J., and E.T. Supervision and funding of plant material engineering, collection, and associated analyses – J.T.B. and A.A.T.J. Supervision and funding of the in vivo experiment and associated analyses – E.T. All authors helped shape the research and reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. 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Muscle Glycogen: Comparison of Iodine Binding and Enzyme Digestion Assays and Application to Meat Samples. \u003cem\u003eMeat Science\u003c/em\u003e\u003cstrong\u003e20\u003c/strong\u003e, 167-177 (1987). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4631411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4631411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Iron (Fe) and zinc (Zn) deficiencies affect over two billion people globally. Biofortification of bread wheat (Triticum aestivum), a crop that supplies approximately 20% of calories and protein consumed by humans worldwide, represents a sustainable strategy for increasing micronutrient intakes. We employed constitutive overexpression (OE) of an endogenous nicotianamine synthase gene (TaNAS2A) in bread wheat cultivar Gladius to increase biosynthesis of the metal-chelating molecule nicotianamine (NA). Field evaluation of three independent OE-TaNAS2A events found normal growth and consistently increased NA concentration in whole wheat flour relative to controls. Extracts prepared from whole wheat flours were functionally characterized in vivo (Gallus gallus) using the intraamniotic administration approach and alterations in markers of Fe and Zn transport, inflammation, and intestinal functionality and morphology were observed in treatment groups that received OE-TaNAS2A extracts.","manuscriptTitle":"Constitutive overexpression of a nicotianamine synthase gene in bread wheat and in vivo assessment of iron and zinc bioavailability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 14:37:58","doi":"10.21203/rs.3.rs-4631411/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b77f8a1-74cf-466d-8605-4e29202cfa4c","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34885009,"name":"Biological sciences/Plant sciences"},{"id":34885010,"name":"Health sciences/Biomarkers"},{"id":34885011,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2024-12-16T07:24:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-24 14:37:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4631411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4631411","identity":"rs-4631411","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}