Energy-dispersive X-ray fluorescence analysis of zinc and iron concentration in rice and pearl millet grain

Background and aims

Rice (Oryza sativa L.) and pearl millet (Pennisetum glaucum L.) biofortification breeding programs require accurate and convenient methods to identify nutrient dense genotypes. The aim of this study was to investigate energy-dispersive X-ray fluorescence spectrometry (EDXRF) for the measurement of zinc (Zn) and iron (Fe) concentration in whole grain rice and pearl millet.

Methods

Grain samples were obtained from existing biofortification breeding programs. Reference Zn and Fe concentrations obtained by inductively-coupled plasma-optical emission spectroscopy (ICP-OES) were used to calibrate the EDXRF instrument. Calibration was performed with 24 samples and separate calibrations were developed for rice and pearl millet. To validate calibrations, EDXRF analyses were conducted on an additional 40 samples of each species.

Results

EDXRF results were highly correlated with ICP-OES values for both Zn and Fe in both species (r²?=?0.79 to 0.98). EDXRF predicted Zn and Fe in rice to within 1.9 and 1.6?mg?kg^?1 of ICP-OES values, and Zn and Fe in pearl millet to within 7.6 and 12.5?mg?kg^?1 of ICP-OES values, at a 95% confidence level.

Conclusion

EDXRF offers a convenient, economical tool for screening Zn and Fe concentration in rice and pearl millet biofortification breeding programs.     

Quantitative trait loci analysis of zinc efficiency and grain zinc concentration in wheat using whole genome average interval mapping

Zinc (Zn) deficiency is a widespread problem which reduces yield and grain nutritive value in many cereal growing regions of the world. While there is considerable genetic variation in tolerance to Zn deficiency (also known as Zn efficiency), phenotypic selection is difficult and would benefit from the development of molecular markers. A doubled haploid population derived from a cross between the Zn inefficient genotype RAC875-2 and the moderately efficient genotype Cascades was screened in three experiments to identify QTL linked to growth under low Zn and with the concentrations of Zn and iron (Fe) in leaf tissue and in the grain. Two experiments were conducted under controlled conditions while the third examined the response to Zn in the field. QTL were identified using an improved method of analysis, whole genome average interval mapping. Shoot biomass and shoot Zn and Fe concentrations showed significant negative correlations, while there were significant genetic correlations between grain Zn and Fe concentrations. Shoot biomass, tissue and grain Zn concentrations were controlled by a number of genes, many with a minor effect. Depending on the traits and the site, the QTL accounted for 12–81% of the genetic variation. Most of the QTL linked to seedling growth under Zn deficiency and to Zn and Fe concentrations were associated with height genes with greater seedling biomass associated with lower Zn and Fe concentrations. Four QTL for grain Zn concentration and a single QTL for grain Fe concentration were also identified. A cluster of adjacent QTL related to the severity of symptoms of Zn deficiency, shoot Zn concentration and kernel weight was found on chromosome 4A and a cluster of QTL associated with shoot and grain Fe concentrations and kernel weight was found on chromosome 3D. These two regions appear promising areas for further work to develop markers for enhanced growth under low Zn and for Zn and Fe uptake. Although there was no significant difference between the parents, the grain Zn concentration ranged from 29 to 43 mg kg^?1 within the population and four QTL associated with grain Zn concentration were identified. These were located on chromosomes 3D, 4B, 6B and 7A and they described 92% of the genetic variation. Each QTL had a relatively small effect on grain Zn concentration but combining the four high Zn alleles increased the grain Zn by 23%. While this illustrates the potential for pyramiding genes to improve grain Zn, breeding for increased grain Zn concentration requires identification of individual QTL with large effects, which in turn requires construction and testing of new mapping populations in the future.     

Quantitative trait loci mapping reveals pleiotropic effect for grain iron and zinc concentrations in wheat

Malnutrition because of the deficiency of minerals such as iron (Fe) and zinc (Zn) afflicts over 2 billion people worldwide. Wheat is a major staple crop, providing 20% of dietary energy and protein consumption worldwide. Breeding wheat with elevated levels of grain Zn and Fe concentrations (GZn and GFe) represents a significant opportunity to increase the intake of these micronutrients for the resource poor people who depend on it as a major source of dietary energy. Synthetic hexaploid wheats (SHWs) have large genetic variation for GZn and GFe, which can be exploited for developing wheat varieties with higher concentrations of these minerals. The objective of this study was to localise genomic regions associated with GZn and GFe, thousand kernel weight (TKW) and test weight (TW) in a mapping population derived from the cross of Seri M82 and the SHW CWI76364. Major quantitative trait loci (QTL) on chromosome 4BS were detected for GZn and GFe; the QTL explained up to 19.6% of the total phenotypic variation for GZn and showed pleiotropic effects on GFe. This indicates that simultaneous improvement of GZn and GFe is feasible. Three and five QTL for TW and TKW were detected, respectively. One of the QTL for TKW was also located on chromosome 4BS. Positive correlations between plant height and GZn/GFe were observed. The 4BS QTL is of great interest for breeding biofortified wheat by means of marker‐assisted selection.     

Distribution of iron, cobalt, zinc and selenium in macrofungi

Samples of wild growing ectomycorrhizal and terrestrial saprobic macrofungi (mushrooms) were collected from unpolluted areas and analyzed for their iron, cobalt, zinc and selenium content. Trace elements were determined using long-term instrumental neutron activation analysis. In total, 217 samples, including 87 species of ectomycorrhizal fungi and 43 species of terrestrial saprobes, were examined. Distribution of trace element contents in ectomycorrhizal and saprobic macrofungi was investigated; results are thoroughly compared with previously published data. Doubtful literature data and ability of macrofungi to accumulate/concentrate investigated elements are discussed. Hygrophoropsis aurantiaca was found to concentrate Fe and Russula atropurpurea was confirmed as an effective Zn-accumulating species. Distribution of Se in ectomycorrhizal species was obviously different from that in saprobic species; selenium contents were higher in saprobic species (mostly above 2 ppm).     

Spatial X-ray fluorescence micro-imaging of minerals in grain tissues of wheat and related genotypes

Main conclusion

Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops.

Abstract

Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.     

Seed priming with iron and zinc in bread wheat: effects in germination,mitosis and grain yield

Currently, the biofortification of crops like wheat with micronutrients such as iron (Fe) and zinc (Zn) is extremely important due to the deficiencies of these micronutrients in the human diet and in soils. Agronomic biofortification with Fe and Zn can be done through different exogenous strategies such as soil application, foliar spraying, and seed priming. However, the excess of these micronutrients can be detrimental to the plants. Therefore, in the last decade, a high number of studies focused on the evaluation of their phytotoxic effects to define the best strategies for biofortification of bread wheat. In this study, we investigated the effects of seed priming with different dosages (1 mg L^?1 to 8 mg L^?1) of Fe and/or Zn in germination, mitosis and yield of bread wheat cv. ‘Jordão’ when compared with control. Overall, our results showed that: micronutrient dosages higher than 4 mg L^?1 negatively affect the germination; Fe and/or Zn concentrations higher than 2 mg L^?1 significantly decrease the mitotic index and increase the percentage of dividing cells with anomalies; treatments performed with 8 mg L^?1 of Fe and/or 8 mg L^?1 Zn caused negative effects in germination, mitosis and grain yield. Moreover, seed priming with 2 mg L^?1 Fe?+?2 mg L^?1 Zn has been shown to be non-cytotoxic, ensuring a high rate of germination (80%) and normal dividing cells (90%) as well as improving tillering and grain yield. This work revealed that seed priming with Fe and Zn micronutrients constitutes a useful and alternative approach for the agronomic biofortification of bread wheat.     

Trends and annual fluctuations in selenium concentrations in wheat grain

Selenium concentrations were determined in grain from four varieties of winter and spring wheat grown in the same area during 1918–1979 and 1930–1980, respectively. Concentrations in the four longest series tended to decrease over time. The annual variation, expressed as the coefficient of variation, ranged between 51%–62% for the four investigated series. According to regression calculations, an additional 100 mm of precipitation during the growing season (May-August) would lead to a 7.2 ng Se g^–1 increase in the Se concentration of spring wheat grain. For winter wheat grain the calculated increase was not significant. No correlation was found between grain yield and grain Se concentration. Although there was a significant difference in grain Se concentrations between spring wheat varieties. there was no indication that older varieties differed from newer ones in this regard.     

Selenium distribution in wheat grain, and the effect of postharvest processing on wheat selenium content

Selenium (Se) is an essential micronutrient for animals and humans, and wheat is a major dietary source of this element. It is improtant that postharvest processing losses of grain Se are minimized. This study, using grain dissection, milling with a Quadrumat mill, and baking and toasting studies, investigated the distribution of Se and other mineral nutrients in wheat grain and the effect of postharvest processing on their retention. The dissection study, although showing Se concentration to be highest in the embryo, confirmed (along with the milling study) previous findings that Se (which occurs mostly as selenomethionine in wheat grain) and S are more evenly distributed throughout the grain when compared to other mineral nutrients, and hence, lower proportions are removed in the milling residue. Postmilling processing did not affect Se concentration or content of wheat products in this study. No genotypic variability was observed for grain distribution of Se in the dissection and milling studies, in contrast to Cu, Fe., Mn, and Zn. This variability could be exploited in breeding for higher proportions of these nutrients in the endosperm to make white flour more nutritious. Further research could include grain dissection and milling studies using larger numbers of cultivars that have been grown together and a flour, extraction rate of around 70%     

Biofortification of iron and zinc in rice and wheat

Iron and zinc are critical micronutrients for human health. Approximately two billion people suffer from iron and zinc deficiencies worldwide, most of whom rely on rice (Oryza sativa) and wheat (Triticum aestivum) as staple foods. Therefore, biofortifying rice and wheat with iron and zinc is an important and economical approach to ameliorate these nutritional deficiencies. In this review, we provide a brief introduction to iron and zinc uptake, translocation, storage, and signaling pathways in rice and wheat. We then discuss current progress in efforts to biofortify rice and wheat with iron and zinc. Finally, we provide future perspectives for the biofortification of rice and wheat with iron and zinc.     

Comparison of X-ray fluorescence and optical fluorescence measured behind scattering layers as a basis for X-ray fluorescence endoscopy.

Recent developments in the technology of capillary-fiber optics suitable for X-rays in the range of approximately 4-10keV point to the possible realization of endoscopes applicable in X-ray fluorescence analysis. A general problem is the determination of scattering and absorption processes with consideration to tissue optics, X-ray scattering and X-ray absorption in a diagnostic partial volume. Therefore comparative investigations were performed in order to answer these questions. Zinc-oxide nanoparticles configured as single particles and ZnO clusters provided the fluorescence source in cell layers. An artificial scattering material was employed, which closely approximated the tissue optical conditions and the X-ray optical application conditions in possible diagnostic situations. As a result imaging of spatially resolved X-ray contrasts was better than adequate optical fluorescence imaging by approximately one magnitude. Hence a very important precondition for realizing X-ray fluorescence endoscopy is fulfilled.     

Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as (48)SO(+). Simultaneously, Fe was measured as (72)FeO(+), P as (47)PO(+), and Zn as (66)Zn(+), enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP(6)) was the main Fe binding ligand, with the stoichiometry Fe(4)(IP(6))(18). When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.     

Plasma manganese, selenium, zinc, copper, and iron concentrations in patients with schizophrenia

A number of essential trace elements play a major role in various metabolic pathways. Selenium (Se), manganese (Mn), copper (Cu), zinc (Zn), and iron (Fe) are essential trace elements that have been studied in many diseases, including autoimmune, neurological, and psychiatric disorders. However, the findings of previous research on the status of trace elements in patients with schizophrenia have been controversial. We studied these elements in patients with a DSM-IV diagnosis of schizophrenia and compared them with sex- and age-matched healthy controls. Plasma Cu concentrations were significantly higher (p<0.01) and Mn and Fe concentrations were lower (p<0.05 and p<0.05, respectively) in schizophrenic patients than in controls. Se and Zn concentrations and protein levels did not differ between patients and healthy controls. These observations suggest that alterations in essential trace elements Mn, Cu, and Fe may play a role in the pathogenesis of schizophrenia. However, findings from trace element levels in schizophrenia show a variety of results that are difficult to interpret.     

Floral analysis as a tool to diagnose iron chlorosis in orange trees

A three-year field experiment was conducted in a commercial orange grove [Citrus sinensis (L.) Osb. cv. `Valencia late' grafted on Citrange Troyer] established on a calcareous soil in the south of Portugal, to investigate if flower analysis could be used to diagnose lime-induced iron chlorosis. In April, during full bloom, flowers and leaves were collected from 20 trees. Leaf samples were again collected from the same trees in May, June, July and August. Total chlorophyll was estimated in all the leaves sampled for foliar analysis, using a SPAD-502 apparatus. Leaves and flowers were analysed for N, P, K, Ca, Mg, Fe, Zn, Mn and Cu. Principal Component Analysis was used to evaluate the variation of nutrient concentrations in flowers, and linear regressions were established between these and the chlorophyll content of leaves 90 days after full bloom. Evaluation of the best-fit equation was carried out using separate data obtained from other groves. Variation in the pattern of floral mineral composition in the flowers showed contrasts between the increase in N, P and K and that of Ca, Fe and Zn, while the concentration of Mg, Mn and Ca varied synchronously. The ratio of Mg:Zn in flowers explained about half of the variation of chlorophyll in leaves later in the season. A ratio below 100 indicated that trees would develop iron chlorosis, while with a ratio above 200 leaves would remain green. An early prognosis of iron chlorosis based on floral analysis can benefit growers, since it allows them to apply treatments in time to prevent loss of fruit yield and quality due to iron chlorosis.     

Substitutions of 2S and 7U chromosomes of Aegilops kotschyi in wheat enhance grain iron and zinc concentration

Biofortification through genetic manipulation is the best approach for improving micronutrient content of the staple food crops to alleviate hidden hunger, namely, the deficiency of Fe and Zn affecting more than two billion people worldwide. An interspecific hybridization was made between T. aestivum line Chinese Spring (CS) and Aegilops kotschyi accession 3790 selected for high grain iron and zinc concentration. The CS × Ae. kotschyi F₁ hybrid with low chromosome pairing was highly male and female sterile. This was backcrossed with wheat cultivars to get seed set. The selfed BC₁F₁ and BC₂F₁ plants with high grain iron and zinc concentration were selected in subsequent generations. The selected derivatives showed 60–136% enhanced grain iron and zinc concentration and 50–120% increased iron and zinc content per seed as compared to the recipient wheat cultivars. Thirteen cytologically stable, fertile and agronomically superior plants with high grain iron and zinc concentrations were selected for molecular characterization. The application of anchored wheat SSR markers, transferable to Ae. kotschyi, to the high grain iron and zinc containing derivatives indicated introgression of group 2 and group 7 chromosomes of Ae. kotschyi. GISH and FISH analysis of some derivatives confirmed the substitution of chromosomes 2S and 7U for their homoeologues of the A genome, suggesting that some of the genes controlling high grain micronutrient content in the Ae. kotschyi accession are on these chromosomes.     

Iron and zinc complexation in wild-type and ferritin-expressing wheat grain: implications for mineral transport into developing grain

We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells and in modified aleurone cells in the transfer region of the grain: iron is coordinated octahedrally by six oxygen atoms and fewer than two phosphorous atoms. Zinc is coordinated tetrahedrally by four oxygen atoms and approximately 1.5 phosphorus atoms in an asymmetric coordination shell. We also present evidence of modified complexation of both metals in transgenic grain overexpressing wheat ferritin. For zinc, there is a consistent doubling of the number of complexing phosphorus atoms. Although there is some EXAFS evidence for iron phytate in ferritin-expressing grain, there is also evidence of a structure lacking phosphorus. This change may lead to an excess of phosphorus within the storage regions of grain, and in turn to the demonstrated increased association of phosphorus with zinc in ferritin-expressing grains. Derivative X-ray absorption spectra also suggest that mineral complexation in the transfer region of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain.     

Chromium,iron, selenium,and zinc levels in serum from preschool children in central Taiwan

The elemental levels of chromium, iron, selenium, and zinc in the sera from 81 preschool children (3-6 yr old) who lived in central Taiwan were determined. One-half of them, at 12 kindergartens in two metropolitan precincts, lived in the Taichung city (TCPC) and the rest lived in 10 urban townships (TUTPC), which had been randomly selected. A blood sample was collected from each subject; sera were freeze-dried, and chromium, iron, selenium, and zinc were measured using instrumental neutron activation analysis (INAA). Results were considered in relation to environmental conditions and the sex and age of the preschool children. The mean concentrations of zinc serum in the TCPC group were statistically significantly higher than those of TUTPC group (p<0.01). The iron sera from girls are higher than those of boys, in both TCPC and TUTPC groups, and show a statistically significant difference (p<0.05) in the TUTPC group. In the TCPC group, Cr contents were positively correlated with age. Elemental concentrations of sera were compared across ages and country.     

InterPro as a new tool for whole genome analysis. A comparative analysis of Mycobacterium tuberculosis, Bacillus subtilis and Escherichia coli as a case study

InterPro was developed as a new integrated documentation resource for protein families, domains and functional sites to rationalize the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects and has applications in computational functional classification of newly determined sequences lacking biochemical characterization and in comparative genome analysis. InterPro contains over 3500 entries, with more than 1000000 hits in SWISS-PROT and TrEMBL. The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. InterPro was used for whole proteome analysis of the pathogenic microorganism, Mycobacterium tuberculosis, and comparison with the predicted protein coding sequences of the complete genomes of Bacillus subtilis and Escherichia coli. 64.8% of the M. tuberculosis proteins in the proteome matched InterPro entries, and these could be classified according to function. The comparison with B. subtilis and E. coli provided information on the most common protein families and domains, and the most highly represented families in each organism. InterPro thus provides a useful tool for global views of whole proteomes and their compositions.