Multi-year field evaluation of nicotianamine biofortified bread wheat.

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

Genetic control of iron bioavailability is independent from iron concentration in a diverse winter wheat mapping population.

BACKGROUND: Anemia is thought to affect up to 1.6 billion people worldwide. One of the major contributors to low iron (Fe) absorption is a higher proportion of cereals compared to meats and pulse crops in people's diets. This has now become a problem in both the developed and developing world, as a result of both modern food choice and food availability. Bread wheat accounts for 20 % of the calories consumed by humans and is an important source of protein, vitamins and minerals meaning it could be a major vehicle for bringing more bioavailable Fe into the diet. RESULTS: To investigate whether breeding for higher concentrations of Fe in wheat grains could help increase Fe absorption, a multiparent advanced generation intercross (MAGIC) population, encompassing more than 80 % of UK wheat polymorphism, was grown over two seasons in the UK. The population was phenotyped for both Fe concentration and Fe bioavailability using an established Caco-2 cell bioassay. It was found that increasing Fe concentrations in the grains was not correlated with higher Fe bioavailability and that the underlying genetic regions controlling grain Fe concentrations do not co-localise with increased Fe absorption. Furthermore, we show that phytate concentrations do not correlate with Fe bioavailability in our wheat population and thus phytate-binding is insufficient to explain the lack of correlation between Fe bioavailability and Fe concentrations in the wheat grain. Finally, we observed no (Fe bioavailability) or low (Fe concentration) correlation between years for these traits, confirming that both are under strong environmental influence. CONCLUSIONS: This suggests that breeders will have to select not only for Fe concentrations directly in grains, but also increased bioavailability. However the use of numerous controls and replicated trials limits the practicality of adoption of screening by Caco-2 cells by many breeders.

Iron Concentrations in Biofortified Beans and Nonbiofortified Marketplace Varieties in East Africa Are Similar.

BACKGROUND: The predominant bean iron (Fe) biofortification approach is to breed for high Fe concentration and assumes the average Fe concentration is 50 µg/g. This approach also assumes that a 40 µg/g increase is sustainable and Fe bioavailability will not decrease to negate the increase in Fe. OBJECTIVE: The overall objective was to determine if bean Fe biofortification via breeding for high Fe is producing beans with higher Fe concentration relative to nonbiofortified lines found in the East Africa marketplace. METHODS: Seventy-six marketplace samples (East Africa Marketplace Collection; EAMC), and 154 genotypes known to be representative of the marketplace were collected from breeders in the Pan-Africa Bean Research Alliance (designated the East Africa Breeder Collection; EABC). Within the EAMC and EABC were 18 and 35 samples, respectively, that were released as biofortified lines. All samples were measured for Fe concentration. The Caco-2 cell bioassay assessed Fe bioavailability of the EAMC. Biofortified versus nonbiofortified samples were compared by the appropriate t-test or ANOVA. RESULTS: The Fe concentration of the 58 nonbiofortified EAMC lines was (mean ± SD [range]) 71 ± 9 µg/g (52-93 µg/g) which did not differ significantly from the 18 biofortified EAMC varieties (71 ± 11 µg/g [55-94 µg/g]). The Fe concentration of the 119 nonbiofortified EABC varieties was 66 ± 7 µg/g (51-90 µg/g) which was significantly different (P < 0.0001) from the 35 EABC biofortified lines (73 ± 9 µg/g [60-91 µg/g]). However, the EABC biofortified lines were not different from the nonbiofortified EAMC samples. In the Caco-2 cell bioassay, biofortified EAMC varieties did not deliver more Fe compared with nonbiofortified EAMC varieties. CONCLUSIONS: The assumptions of the high Fe bean biofortification approach are not met in the East African marketplace. Iron concentration and bioavailability measurement indicate the biofortified bean varieties are providing no additional dietary Fe.

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

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up-regulate biosynthesis of two low molecular weight metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - that play key roles in metal transport and nutrition. The CE-OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X-ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field-grown CE-OsNAS2 grain and positively correlated with NA and DMA concentrations.

Seasonal trends of nutrient intake in rainforest communities of north-eastern Madagascar.

OBJECTIVE: We collected dietary records over the course of nine months to comprehensively characterize the consumption patterns of Malagasy people living in remote rainforest areas of north-eastern Madagascar. DESIGN: The present study was a prospective longitudinal cohort study to estimate dietary diversity and nutrient intake for a suite of macronutrients, micronutrients and vitamins for 152 randomly selected households in two communities. SETTING: Madagascar, with over 25 million people living in an area the size of France, faces a multitude of nutritional challenges. Micronutrient-poor staples, especially rice, roots and tubers, comprise nearly 80 % of the Malagasy diet by weight. The remaining dietary components (including wild foods and animal-source foods) are critical for nutrition. We focus our study in north-eastern Madagascar, characterized by access to rainforest, rice paddies and local agriculture. PARTICIPANTS: We enrolled men, women and children of both sexes and all ages in a randomized sample of households in two communities. RESULTS: Although the Household Dietary Diversity Score and Food Consumption Score reflect high dietary diversity, the Minimum Dietary Diversity-Women indicator suggests poor micronutrient adequacy. The food intake data confirm a mixed nutritional picture. We found that the median individual consumed less than 50 % of his/her age/sex-specific Estimated Average Requirement (EAR) for vitamins A, B12, D and E, and Ca, and less than 100 % of his/her EAR for energy, riboflavin, folate and Na. CONCLUSIONS: Malnutrition in remote communities of north-eastern Madagascar is pervasive and multidimensional, indicating an urgent need for comprehensive public health and development interventions focused on providing nutritional security.

Single Varietal Dry Bean (Phaseolus vulgaris L.) Pastas: Nutritional Profile and Consumer Acceptability.

Dry beans (Phaseolus vulgaris L.) are a nutrient dense food rich in protein, dietary fiber, minerals, and folate. Consumption of dry beans is relatively low in the United States and this may be due in part to the lack of diversity in bean products beyond whole seeds. Products that incorporate beans in new forms, such as flours, offer the potential to increase dry bean utilization. In this study whole dry beans were milled into flour and used to make gluten free fresh pastas. Six bean varieties each from a different market class (white kidney, navy, otebo, cranberry, dark red kidney and black) were made into pasta. Their consumer appeal was compared to fresh wheat pasta and their nutritional value was compared to fresh wheat pasta and whole boiled beans. The dry bean pastas were nutritionally superior to wheat pasta with higher protein, ash, resistant starch and protein digestibility corrected amino acid score (PDCAAS) as well as lower total starch content. While consumers preferred the flavor, texture and appearance of the wheat pasta to the dry bean pasta, 36% of participants said they would definitely or probably purchase the dry bean pastas from the light colored beans. There was some loss of nutritional value of bean pasta vs. whole boiled beans but this can mostly be attributed to the bean pasta being 90% bean. These results suggest that single variety fresh dry bean pastas have commercial potential in the U.S. as healthy gluten free pasta options.

Iron Absorption from an Intrinsically Labeled Lentil Meal Is Low but Upregulated in Women with Poor Iron Status.

BACKGROUND: Low iron absorption from important staple foods may contribute to iron deficiency in developing countries. To date, few studies have examined the iron bioavailability of pulse crops as commonly prepared and consumed by humans. OBJECTIVE: The objectives were to characterize the iron absorption from a test meal of intrinsically labeled (57)Fe lentils prepared as dal, to compare the bioavailability of iron from (57)Fe in dal with that observed for a reference dose of (58)Fe as ferrous sulfate, and to assess associations between iron absorption and iron status indicators. METHODS: This crossover study included 19 nonpregnant women (n = 6 anemic; hemoglobin: <12.0 g/dL) who consumed 2 test meals on consecutive days in a counter-balanced order, ferrous sulfate (7 mg FeSO4 plus 1 mg (58)Fe) and 330 g dal (lentils enriched to 85.1% with (57)Fe, 8 mg native (57)Fe). Iron absorption was determined by analyzing blood samples taken 14 d after dosing with the use of magnetic sector thermal ionization mass spectrometry. RESULTS: We found that the mean iron absorption from the dal was 2.20% ± 3.40% and was significantly lower than the 23.6% ± 13.2% observed from the same iron load given as ferrous sulfate (P < 0.001). Absorption of non-heme iron from dal and from ferrous sulfate was inversely associated with serum ferritin (SF; r = -0.50, P = 0.05 and r = -0.81, P < 0.001, respectively) and serum hepcidin (r = -0.45, P = 0.05 and r = -0.60, P = 0.007, respectively). Anemic women absorbed more iron from either source (1.20% from dal, P = 0.10; 18.3% from ferrous sulfate, P = 0.001) compared with women who were iron replete. CONCLUSIONS: Iron absorption from the dal was low overall but upregulated in anemic women. Both SF and hepcidin were inversely associated with iron absorption from both a supplemental and a food-based non-heme iron source in nonanemic and anemic women.

Higher iron pearl millet (Pennisetum glaucum L.) provides more absorbable iron that is limited by increased polyphenolic content.

BACKGROUND: Our objective was to compare the capacity of iron (Fe) biofortified and standard pearl millet (Pennisetum glaucum L.) to deliver Fe for hemoglobin (Hb)-synthesis. Pearl millet (PM) is common in West-Africa and India, and is well adapted to growing areas characterized by drought, low-soil fertility, and high-temperature. Because of its tolerance to difficult growing conditions, it can be grown in areas where other cereal crops, such as maize, would not survive. It accounts for approximately 50% of the total world-production of millet. Given the widespread use of PM in areas of the world affected by Fe-deficiency, it is important to establish whether biofortified-PM can improve Fe-nutriture. METHODS: Two isolines of PM, a low-Fe-control ("DG-9444", Low-Fe) and biofortified ("ICTP-8203 Fe",High-Fe) in Fe (26 µg and 85 µg-Fe/g, respectively) were used. PM-based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (Fe concentrations were 22.1±0.52 and 78.6±0.51 µg-Fe/g for the Low-Fe and High-Fe diets, respectively). For 6-weeks, Hb, feed-consumption and body-weight were measured (n = 12). RESULTS: Improved Fe-status was observed in the High-Fe group, as suggested by total-Hb-Fe values (15.5±0.8 and 26.7±1.4 mg, Low-Fe and High-Fe respectively, P<0.05). DMT-1, DcytB, and ferroportin mRNA-expression was higher (P<0.05) and liver-ferritin was lower (P>0.05) in the Low-Fe group versus High-Fe group. In-vitro comparisons indicated that the High-Fe PM should provide more absorbable-Fe; however, the cell-ferritin values of the in-vitro bioassay were very low. Such low in-vitro values, and as previously demonstrated, indicate the presence of high-levels of polyphenolic-compounds or/and phytic-acid that inhibit Fe-absorption. LC/MS-analysis yielded 15 unique parent aglycone polyphenolic-compounds elevated in the High-Fe line, corresponding to m/z = 431.09. CONCLUSIONS: The High-Fe diet appeared to deliver more absorbable-Fe as evidenced by the increased Hb and Hb-Fe status. Results suggest that some PM varieties with higher Fe contents also contain elevated polyphenolic concentrations, which inhibit Fe-bioavailability. Our observations are important as these polyphenols-compounds represent potential targets which can perhaps be manipulated during the breeding process to yield improved dietary Fe-bioavailability. Therefore, the polyphenolic and phytate profiles of PM must be carefully evaluated in order to further improve the nutritional benefit of this crop.

The effect of wheat prebiotics on the gut bacterial population and iron status of iron deficient broiler chickens.

BACKGROUND: Currently, there is a lot of interest in improving gut health, and consequently increasing Fe absorption, by managing the colonic microbial population. This is traditionally done by the consumption of probiotics, live microbial food supplements. However, an alternative, and often very effective approach, is the consumption of food ingredients known as prebiotics. Fructans and arabinoxylans are naturally occurring non-digestible oligosaccharides in wheat that exhibit prebiotic properties and may enhance intestinal iron (Fe) absorption. The aim of this study was to assess the effect of prebiotics from wheat on Fe bioavailability in vitro (Caco-2 cells) and in vivo (broiler chickens, Gallus gallus). METHODS: In the current study, the effect of intra-amniotic administration of wheat samples extracts at 17 d of embryonic incubation on the Fe status and possible changes in the bacterial population in intestinal content of broiler hatchlings were investigated. A group of 144 eggs were injected with the specified solution (1 ml per egg) into the amniotic fluid. Immediately after hatch (21 d) and from each treatment group, 10 chicks were euthanized and their small intestine, liver and cecum were removed for relative mRNA abundance of intestinal Fe related transporters, relative liver ferritin amounts and bacterial analysis of cecal content, respectively. RESULTS: The in vivo results are in agreement with the in vitro observations, showing no differences in the hatchling Fe status between the treatment groups, as Fe bioavailability was not increased in vitro and no significant differences were measured in the intestinal expression of DMT1, Ferroportin and DcytB in vivo. However, there was significant variation in relative amounts of bifidobacteria and lactobacilli in the intestinal content between the treatments groups, with generally more bifidobacteria being produced with increased prebiotic content. CONCLUSIONS: In this study we showed that prebiotics naturally found in wheat grains/bread products significantly increased intestinal beneficial bacterial population in Fe deficient broiler chickens. With this short-term feeding trial we were not able to show differences in the Fe-status of broilers. Nevertheless, the increase in relative amounts of bifidobacteria and lactobacilli in the presence of wheat prebiotics is an important finding as these bacterial populations may affect Fe bioavailability in long-term studies.

Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.).

BACKGROUND: Our objective was to determine if a biofortified variety of black bean can provide more bioavailable-iron (Fe) than a standard variety. Two lines of black beans (Phaseolus-vulgaris L.), a standard (DOR500; 59µg Fe/g) and biofortified (MIB465; 88µg Fe/g) were used. The DOR500 is a common commercial variety, and the MIB465 is a line developed for higher-Fe content. Given the high prevalence of Fe-deficiency anemia worldwide, it is important to determine if Fe-biofortified black beans can provide more absorbable-Fe. METHODS: Black bean based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (dietary Fe-concentrations were 39.4±0.2 and 52.9±0.9 mg/kg diet, standard vs. biofortified, respectively). Birds (n=14) were fed the diets for 6-weeks. Hemoglobin-(Hb), liver-ferritin and Fe-related transporter/enzyme gene-expression were measured. Hemoglobin-maintenance-efficiency and total-body-Hb-Fe values were used to estimate Fe-bioavailability. RESULTS: Hemoglobin-maintenance-efficiency values were higher (P<0.05) in the group consuming the standard-Fe beans on days 14, 21 and 28; indicating a compensatory response to lower dietary-Fe. Final total-Hb-Fe body content was higher in the biofortified vs. the standard group (26.6±0.9 and 24.4±0.8 mg, respectively; P<0.05). There were no differences in liver-ferritin or in expression of DMT-1, Dcyt-B, and ferroportin. In-vitro Fe-bioavailability assessment indicated very low Fe-bioavailability from both diets and between the two bean varieties (P>0.05). Such extremely-low in-vitro Fe-bioavailability measurement is indicative of the presence of high levels of polyphenolic-compounds that may inhibit Fe-absorption. High levels of these compounds would be expected in the black bean seed-coats. CONCLUSIONS: The parameters of Fe-status measured in this study indicate that only a minor increase in absorbable-Fe was achieved with the higher-Fe beans. The results also raise the possibility that breeding for increased Fe-concentration elevated the levels of polyphenolic-compounds that can reduce bean Fe-bioavailability, although the higher levels of polyphenolics in the higher-Fe beans may simply be coincidental or an environmental effect. Regardless, Fe-biofortified beans remain a promising vehicle for increasing intakes of bioavailable-Fe in human populations that consume high levels of these beans as a dietary staple, and the bean polyphenol profile must be further evaluated and modified if possible in order to improve the nutritional quality of higher-Fe beans.

Differences in relative iron bioavailability in traditional Bangladeshi meal plans.

BACKGROUND: Iron deficiency is the most common nutrient deficiency worldwide. Large intakes of micronutrient-poor staple crops, coupled with low intakes of highly bioavailable dietary iron, are a major cause of this deficiency. OBJECTIVE: To examine the concentration and relative bioavailability of iron in several models (n = 23) of traditional Bangladeshi meals (rice, lentils/dal, vegetable, and fish), as well as the effect of removal of the lentil seed coat on the nutritional quality of iron. METHODS: The relative bioavailability of iron was assessed by the in vitro/Caco-2 cell culture method, iron concentration by an inductively coupled argon-plasma emission spectrometer (ICAP-ES), and phytic acid concentration by colorimetric assay. The recipes contained 75% to 85% rice, 0% to 15% dal (containing whole or dehulled lentils), 0% to 15% vegetable curry, and 0% to 8% fish. RESULTS: While the iron concentrations of recipes containing dehulled dal were significantly lower than those of recipes containing whole dal (p = .005), seed coat removal doubled relative iron bioavailability and increased phytic acid concentration (p < .001). The addition of fish to the meals had no significant effect on relative iron bioavailability. Iron concentration and relative iron bioavailability were correlated in the recipes containing dehulled dal (r = 0.48, p = .03), but not whole dal (r = -0.047, p = .84). CONCLUSIONS: The total amount of iron absorbed from traditional Bangladeshi meals is dependent upon iron concentration, and dehulling lentils removes inhibitory factors increasing iron uptake but also increases the density of phytic acid in the lentil sample. Thus, along with breeding for high iron concentration and bioavailability (i.e., biofortification), seed coat removal plus measures to lower phytic acid concentrations may be an important strategy to improve the bioavailability of iron in lentils and other pulse crops.

Chemical composition and in vitro iron bioavailability of extruded and open-pan cooked germinated and ungerminated pearl whole millet "Pennisetum glaucum (L.) R. Br."

This study aimed to evaluate the effect of extrusion and of open-pan cooking on whole germinated and non-germinated grains of pearl millet (Pennisetum glaucum L. R. Br.), on its chemical-nutritional composition and in vitro iron bioavailability. The experimental design consisted of three flours: non-germination open-pan cooked millet flour (NGOPCMF), germination open-pan cooked millet flour (GOPCMF), and extrusion cooked millet flour (ECMF). The ECMF increased the carbohydrates, iron, manganese, diosmin, and cyanidin and decreased the total dietary fiber, resistant starch, lipids, and total vitamin E, in relation to NGOPCMF. The GOPCMF increased the lysine and vitamin C and decreased the phytate, lipids, total phenolic, total vitamin E, and riboflavin concentration, in relation to NGOPCMF. Furthermore, germinated cooked millet flour and extruded millet flour improved iron availability in vitro compared to non-germinated cooked millet flour. GOPCMF and ECMF generally preserved the chemical-nutritional composition of pearl millet and improved in vitro iron bioavailability; therefore, they are nutritionally equivalent and can be used to develop pearl millet-based products.

Soil consumed by chacma baboons is low in bioavailable iron and high in clay.

Despite widespread consumption of soil among animals, the role of geophagy in health maintenance remains an enigma. It has been hypothesized that animals consume soil for supplementation of minerals and protection against toxins. Most studies determine only the total elemental composition of soil, which may not reflect the amount of minerals available to the consumer. Our aim was to test these hypotheses by evaluating the bioavailability of iron in soil consumed by chacma baboons, using a technique that simulates digestion and adsorption. Our results indicate that, despite variation in absolute iron concentration of soil samples, actual iron bioavailability was low while clay content was quite high. This suggests that iron supplementation is unlikely to be the primary motivation for geophagy in this population, and that detoxification is a plausible explanation. This study demonstrates that more research on bioavailability and clay composition is needed to determine the role geophagy plays in health maintenance.

High bioavailability iron maize (Zea mays L.) developed through molecular breeding provides more absorbable iron in vitro (Caco-2 model) and in vivo (Gallus gallus).

BACKGROUND: Iron (Fe) deficiency is the most common micronutrient deficiency worldwide. Iron biofortification is a preventative strategy that alleviates Fe deficiency by improving the amount of absorbable Fe in crops. In the present study, we used an in vitro digestion/Caco 2 cell culture model as the guiding tool for breeding and development of two maize (Zea mays L.) lines with contrasting Fe bioavailability (ie. Low and High). Our objective was to confirm and validate the in vitro results and approach. Also, to compare the capacities of our two maize hybrid varieties to deliver Fe for hemoglobin (Hb) synthesis and to improve the Fe status of Fe deficient broiler chickens. METHODS: We compared the Fe-bioavailability between these two maize varieties with the presence or absence of added Fe in the maize based-diets. Diets were made with 75% (w/w) maize of either low or high Fe-bioavailability maize, with or without Fe (ferric citrate). Chicks (Gallus gallus) were fed the diets for 6 wk. Hb, liver ferritin and Fe related transporter/enzyme gene-expression were measured. Hemoglobin maintenance efficiency (HME) and total body Hb Fe values were used to estimate Fe bioavailability from the diets. RESULTS: DMT-1, DcytB and ferroportin expressions were higher (P<0.05) in the "Low Fe" group than in the "High Fe" group (no added Fe), indicating lower Fe status and adaptation to less Fe-bioavailability. At times, Hb concentrations (d 21,28,35), HME (d 21), Hb-Fe (as from d 14) and liver ferritin were higher in the "High Fe" than in the "Low Fe" groups (P<0.05), indicating greater Fe absorption from the diet and improved Fe status. CONCLUSIONS: We conclude that the High Fe-bioavailability maize contains more bioavailable Fe than the Low Fe-bioavailability maize, presumably due to a more favorable matrix for absorption. Maize shows promise for Fe biofortification; therefore, human trials should be conducted to determine the efficacy of consuming the high bioavailable Fe maize to reduce Fe deficiency.

Iron bioavailability of maize (Zea mays L.) after removing the germ fraction.

Maize is a staple food for many communities with high levels of iron deficiency anemia. Enhancing the iron concentrations and iron bioavailability of maize with traditional breeding practices, especially after cooking and processing, could help alleviate iron deficiency in many of these regions. Previous studies on a small number of maize genotypes and maize flour products indicated that degermination (germ fraction removed with processing) could improve the iron bioavailability of maize. This study expanded upon this research by evaluating the iron bioavailability, mineral concentrations, and phytate concentrations of 52 diverse maize genotypes before (whole kernels) and after degermination. Whole and degerminated maize samples were cooked, dried, and milled to produce corn flour. Iron bioavailability was evaluated with an in vitro digestion Caco2 cell bioassay. In 30 of the maize genotypes, bioavailable iron increased when degerminated, thus indicating a higher fractional iron uptake because the iron concentrations decreased by more than 70% after the germ fraction was removed. The remaining 22 genotypes showed no change or a decrease in iron bioavailability after degermination. These results confirm previous research showing that the germ fraction is a strong inhibitory component for many maize varieties. Phytate concentrations in maize flours were greatly reduced with degermination. However, the relationship between phytate concentrations and the iron bioavailability of processed maize flour is complex, acting as either inhibitor or promoter of iron uptake depending on the color of the maize kernels and processing method used to produce flour. Other factors in the maize endosperm fractions are likely involved in the effects of degermination on iron bioavailability, such as vitreous or floury endosperm compositions and the polyphenol content of the bran. This study demonstrates that iron nutrition from maize can be enhanced by selecting genotypes where the inhibitory effect of the bran color and endosperm fraction are relatively low, especially after processing via degermination.

Metacridamides A and B, macrocycles from conidia of the entomopathogenic fungus Metarhizium acridum.

Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A and B, which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A showed cytotoxicity to three cancer lines with IC50's of 6.2, 11.0, and 10.8 µM against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B had an IC50 of 18.2 µM against HepG2/C3A, although it was inactive at 100 µM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

Identifying genes associated with abiotic stress tolerance suitable for CRISPR/Cas9 editing in upland rice cultivars adapted to acid soils.

Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought-prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for gene editing: SUB1A (Submergence 1A), to improve tolerance to flooding, and SPDT (SULTR3;4) (SULTR-like Phosphorus Distribution Transporter), to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild-type (nondesirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system, and specialized reagents were developed for SPDT editing, including vectors targeting the gene and a protoplast transfection transient assay. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P-use efficiency of upland rice grown in acidic soils.

The Common Bean V Gene Encodes Flavonoid 3'5' Hydroxylase: A Major Mutational Target for Flavonoid Diversity in Angiosperms.

The classic V (violet, purple) gene of common bean (Phaseolus vulgaris) functions in a complex genetic network that controls seed coat and flower color and flavonoid content. V was cloned to understand its role in the network and the evolution of its orthologs in the Viridiplantae. V mapped genetically to a narrow interval on chromosome Pv06. A candidate gene was selected based on flavonoid analysis and confirmed by recombinational mapping. Protein and domain modeling determined V encodes flavonoid 3'5' hydroxylase (F3'5'H), a P450 enzyme required for the expression of dihydromyricetin-derived flavonoids in the flavonoid pathway. Eight recessive haplotypes, defined by mutations of key functional domains required for P450 activities, evolved independently in the two bean gene pools from a common ancestral gene. V homologs were identified in Viridiplantae orders by functional domain searches. A phylogenetic analysis determined F3'5'H first appeared in the Streptophyta and is present in only 41% of Angiosperm reference genomes. The evolutionarily related flavonoid pathway gene flavonoid 3' hydroxylase (F3'H) is found nearly universally in all Angiosperms. F3'H may be conserved because of its role in abiotic stress, while F3'5'H evolved as a major target gene for the evolution of flower and seed coat color in plants.

Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/Caco-2 model.

BACKGROUND: Our objective was to compare the capacities of biofortified and standard colored beans to deliver iron (Fe) for hemoglobin synthesis. Two isolines of large-seeded, red mottled Andean beans (Phaseolus vulgaris L.), one standard ("Low Fe") and the other biofortified ("High Fe") in Fe (49 and 71 µg Fe/g, respectively) were used. This commercial class of red mottled beans is the preferred varietal type for most of the Caribbean and Eastern and Southern Africa where almost three quarters of a million hectares are grown. Therefore it is important to know the affect of biofortification of these beans on diets that simulate human feeding studies. METHODS: Maize-based diets containing the beans were formulated to meet the nutrient requirements for broiler except for Fe (Fe concentrations in the 2 diets were 42.9 ± 1.2 and 54.6 ± 0.9 mg/kg). One day old chicks (Gallus gallus) were allocated to the experimental diets (n = 12). For 4 wk, hemoglobin, feed-consumption and body-weights were measured. RESULTS: Hemoglobin maintenance efficiencies (HME) (means ± SEM) were different between groups on days 14 and 21 of the experiment (P < 0.05). Final total body hemoglobin Fe contents were different between the standard (12.58 ± 1.0 mg {0.228 ± 0.01 µmol}) and high Fe (15.04 ± 0.65 mg {0.273 ± 0.01 µmol}) bean groups (P < 0.05). At the end of the experiment, tissue samples were collected from the intestinal duodenum and liver for further analyses. Divalent-metal-transporter-1, duodenal-cytochrome-B, and ferroportin expressions were higher and liver ferritin was lower (P < 0.05) in the standard group vs. the biofortified group. In-vitro analysis showed lower iron bioavailability in cells exposed to standard ("Low Fe") bean based diet. CONCLUSIONS: We conclude that the in-vivo results support the in-vitro observations; biofortified colored beans contain more bioavailable-iron than standard colored beans. In addition, biofortified beans seems to be a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume these beans as a dietary staple. This justifies further work on the large-seeded Andean beans which are the staple of a large-region of Africa where iron-deficiency anemia is a primary cause of infant death and poor health status.