Localization of iron in rice grain using synchrotron X-ray fluorescence microscopy and high resolution secondary ion mass spectrometry

Cereal crops accumulate low levels of iron (Fe) of which only a small fraction (5–10%) is bioavailable in human diets. Extensive co-localization of Fe in outer grain tissues with phytic acid, a strong chelator of metal ions, results in the formation of insoluble complexes that cannot be digested by humans. Here we describe the use of synchrotron X-ray fluorescence microscopy (XFM) and high resolution secondary ion mass spectrometry (NanoSIMS) to map the distribution of Fe, zinc (Zn), phosphorus (P) and other elements in the aleurone and subaleurone layers of mature grain from wild-type and an Fe-enriched line of rice (Oryza sativa L.). The results obtained from both XFM and NanoSIMS indicated that most Fe was co-localized with P (indicative of phytic acid) in the aleurone layer but that a small amount of Fe, often present as “hotspots”, extended further into the subaleurone and outer endosperm in a pattern that was not co-localized with P. We hypothesize that Fe in subaleurone and outer endosperm layers of rice grain could be bound to low molecular weight chelators such as nicotianamine and/or deoxymugineic acid.     

Evaluation of Tissue Dissociation of Durum Wheat Grain (Triticum durum Desf.) Generated by the Milling Process

The three major botanical components (starchy endosperm, aleurone layer and pericarp) of eight durum wheat samples exhibited significantly different compositions and concentrations in phenolic acids. The starchy endosperm, the aleurone layer and the pericarp were respectively characterised by a low content in ferulic acid (FA), a high content intrans -sinapic acid (t -SA), and a high content in ferulic acid dehydrodimers (DHD). These three chemical markers can be exploited to differentiate the three grain botanical parts within milling fractions and to evaluate the milling efficiency, particularly the separation between bran and endosperm. The histological dissociation of the wheat grain generated by the milling process can be investigated further into details using the three phenolic acids markers. A separability index (S i) was proposed in order to quantify the ease of dissociation of endosperm from bran. Differences in S i values between wheat varieties grown under various agricultural conditions demonstrated the relevant variability of this character. The structural and molecular factors implied in the control of tissue dissociation are discussed in details.     

Improved nitrogen status enhances zinc and iron concentrations both in the whole grain and the endosperm fraction of wheat

This study was conducted to assess the role of increasing N supply in enrichment of whole grain and grain fractions, particularly the endosperm, with Zn and Fe in wheat. The endosperm is the most widely consumed part of wheat grain in many countries. Plants were grown in the greenhouse with different soil applications of N and Zn and with or without foliar Zn spray. Whole grain and grain fractions were analyzed for N, P, Zn and Fe. Increased N supply significantly enhanced the Zn and Fe concentrations in all grain fractions. In the case of high Zn supply, increasing N application enhanced the whole grain Zn concentration by up to 50% and the endosperm Zn by over 80%. Depending on foliar Zn supply, high N elevated the endosperm Fe concentration up to 100%. High N also generally decreased the P/Zn and P/Fe molar ratios in whole grain and endosperm. The results demonstrate that improved N nutrition, especially when combined with foliar Zn treatment, is effective in increasing Zn and Fe of the whole grain and particularly the endosperm fraction, at least in the greenhouse, and might be a promising strategy for tackling micronutrient deficiencies in countries where white flour is extensively consumed.     

Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions

To produce safe and healthy whole wheat food products, various grain or bran dry fractionation processes have been developed recently. In order to control the quality of the products and to adapt these processes, it is important to be able to monitor the grain tissue proportions in the different milling fractions produced. Accordingly, a quantitative method based on biochemical markers has been developed for the assessment of grain tissue proportions in grain fractions. Grain tissues that were quantified were the outer pericarp, an intermediate layer (including the outer pericarp, the testa and the hyaline layer), the aleurone cell walls, the aleurone cell contents, the endosperm and the germ, for two grain cultivars (Tiger and Crousty). Grain tissues were dissected by hand and analysed. Biochemical markers chosen were ferulic acid trimer, alkylresorcinols, para-coumaric acid, phytic acid, starch and wheat germ agglutinin, for outer pericarp, intermediate layer, aleurone cell walls, aleurone cell contents, endosperm and germ respectively. The results of tissue quantification by hand dissection and by calculation were compared and the sensitivity of the method was regarded as good (mean relative errors of 4% and 8% for Crousty and Tiger outer layers respectively). The impact of the analytical variability (maximum 13% relative error on coarse bran) was also regarded as acceptable. Wheat germ agglutinin seems to be a promising marker of wheat germ: even if the quantification method was not able to quantify the germ proportions in milling fractions, it was able to classify these fractions according to their germ content. The efficiency of this method was tested, by assessing the grain tissue proportions of fractions exhibiting very different compositions such as flour, bran and aleurone-rich fractions obtained from three different grain or bran dry fractionation processes (conventional milling, debranning process, production of aleurone-rich fractions from coarse bran). By calculation of the composition of the different products generated, it was possible to study the distribution of the different tissues among fractions resulting from the different fractionation processes. This quantitative method is thus a useful tool for the monitoring and improvement of processes, and allows the effects of these processes to be understood and their adaption to reach the objectives.     

小麦胚乳传递细胞发育的结构观察

为从细胞学方面了解小麦产量和品质的形成机制,以扬麦5号为材料,利用光镜和透射电镜观察了小麦颖果发育过程中胚乳传递细胞的结构变化,并探讨了胚乳传递细胞的生理功能。结果表明：（1）胚乳传递细胞是胚乳发育过程中最早分化的细胞类型,它们发生在紧邻胚乳腔的胚乳表层,由位于外侧1~2层糊粉层传递细胞和位于内侧1~2层内胚乳传递细胞构成;（2）颖果发育成熟时,内胚乳传递细胞核衰亡,糊粉层传递细胞核依然完整;（3）胚乳传递细胞发育呈明显的极性,且具时空性;（4）糊粉层传递细胞胞质较浓,富含粗面内质网、线粒体、高尔基体和脂质体;质膜皱褶,在局部区域外翻,形成众多的原生质管;（5）内胚乳传递细胞胞质较稀,液泡化程度较高,富含淀粉体;（6）胚乳传递细胞未加厚以及未形成壁内突的壁区域分布有大量的胞间连丝;（7）胚乳传递细胞中线粒体呈极性分布,即质膜附近线粒体的密度较大。根据胚乳传递细胞的结构特点推测,经胚乳传递细胞的养分输送既可通过质外体途径又可通过共质体途径来完成。     

不同类型专用小麦品种胚乳发育的比较研究

为揭示不同类型专用小麦品种胚乳发育的差异,以强筋小麦皖麦38和弱筋小麦扬麦9号为材料,比较了两者胚乳细胞游离核分裂状况、细胞数目的增殖变化、细胞体积的变化、糊粉层发育、淀粉体和蛋白质体发育等内容,主要结果如下：（1）皖麦38游离核有丝分裂所占比例较大,扬麦9号无丝分裂所占比例较大,且受温度影响较大;（2）胚乳细胞增殖均呈"S"型曲线变化,皖麦38胚乳细胞增殖较快,胚乳体积较大,最终粒重较高;（3）皖麦38胚乳细胞中大淀粉体数目多于扬麦9号,而小淀粉体数目表现却相反;（4）皖麦38与扬麦9号相比较,糊粉层出现时间较晚,而且糊粉层细胞壁较厚,被甲苯胺蓝染色更浓;（5）成熟籽粒中淀粉粒形状有饼形、椭圆形和近圆球形三种。皖麦38胚乳中饼形和椭圆形淀粉粒较多,且相互结合较紧密;扬麦9号饼形和近球形淀粉粒较多,胚乳结构疏松。     

Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy

Grain ultrastructure is of utmost importance when designing grain processing procedures in the food industry. In this study, wheat and barley grain components were localised using optical and Raman microscopy. The optical microscopic analyses were performed using several selective stains to localise β-glucan, protein and starch or autofluorescence to image the ferulic acid and other fluorescing substances. Alternatively, Raman microscopy was applied to localise the grain components without any need for preceding staining or other sample pretreatment. Both methods provided consistent information on the grain structures, illustrating the distribution of polysaccharides, aromatics and protein in endosperm and aleurone layers. In aleurone layers of both barley and wheat, a distinct difference between the anticlinal and periclinal cell walls was observed. The anticlinal cell walls were enriched with aromatic substances which were present in remarkably lower concentrations in the inner periclinal cell walls but for barley, an even higher concentration in the outer periclinal cell wall was observed. In addition, Raman spectroscopy illustrated the detailed distribution of substances across the aleurone cell walls: β-glucan was adjacent to proteins and it was deficient in the middle lamella whereas arabinoxylan was enriched in the outer cell wall layers and middle lamella.     

Spatial distribution of functional components in the starchy endosperm of wheat grains

The starchy endosperm of the mature wheat grain comprises three major cell types, namely sub-aleurone cells, prismatic cells and central cells, which differ in their contents of functional components: gluten proteins, starch, cell wall polysaccharides (dietary fibre) and lipids. Gradients are established during grain development but may be modified during grain maturation and are affected by plant nutrition, particularly nitrogen application, and environmental factors. Although the molecular controls of their formation are unknown, the high content of protein and low content of starch of sub-aleurone cells, compared to the other starchy endosperm cells types, may result from differences in developmental programming related to the cells having a separate origin (from anticlinal division of the aleurone cells). The gradients within the grain may be reflected in differences in the compositions of mill streams, particularly those streams enriched in the central and outer cells of the starchy endosperm, respectively, allowing the production of specialist flours for specific end uses.     

Wheat bran tissue fractionation using biochemical markers

Phenolic acid analysis of hand-isolated outer grain layers and endosperm led to the identification of markers of pericarp and aleurone layers, respectively. A new dehydrotrimer of ferulic acid (DHT) was found to be concentrated in the outer pericarp of wheat bran whereas p-coumaric (p-CA) acid was mainly in the aleurone layer. Phytates were also used as a marker of aleurone layer and starch as a marker of starchy endosperm. Biochemical markers constitute an original method for determining the histological composition of any technological bran fractions. A pin milling process was applied to coarse bran produced by a conventional milling process. Three different fractions (B1, B2 and B3) were obtained by sieving the bran products and then the smallest bran particle fraction (B3) was air-classified to obtain two particle size fractions (B3a and B3b with a D50 of 83 and 7 μm, respectively). The biochemical composition of these fractions was used to calculate the distribution of tissues according to the sieving process. The dissociation behavior of individual bran tissues upon mechanical fractionation was investigated in relation to particle size and discussed according to their mechanical properties.     

Variability in dehydrodiferulic acid composition of durum wheat (Triticum durum Desf.) and distribution in milling fractions

The dehydrodiferulic acid content of different common and durum wheat grains and milling fractions was determined by an HPLC procedure. The 8-O-4′, 5–8′ benzofuran, 5–8′ and 5-5′ dehydrodimers were identified in all samples studied and occurred in decreasing relative amounts, respectively. Durum wheats were twice as concentrated in dimers as common wheats. An important genetic variation for dehydrodiferulic acid content was shown within durum wheat grains, whereas the agronomic conditions had no effect. There was 10 to 20 times more dehydrodiferulic acids in external layers (aleurone, bran) than in the starchy endosperm of durum wheat grains. The content and composition in dimers of the inner endosperm did not vary according to genotypes and cultivation conditions. The ratio of dehydrodimers to monomers of ferulic acid which represented an index of dimerisation, was 1·6 times higher in the external layers of the grain than in the endosperm. No relation was found, however, between the degree of ferulic acid dimerisation and the milling behaviour of durum wheat grains.     

Mineral element distributions in milling fractions of Chinese wheats

Malnutrition related to micronutrient deficiency can create immense economic and societal problems. The objective of this study was to quantify the mineral element concentration distribution in milled fractions, using 43 common wheat (Triticum aestivum L.) cultivars sown in Jinan, China during the 2005–2006 crop season. All 43 cultivars had low Fe (average 28.2 mg Kg⁻¹) and Zn (28.6 mg Kg⁻¹) concentrations, and wide ranges of variation for mineral element concentrations. Highly significant effects among milling fractions and cultivars on all traits were observed, with fraction effect being the larger. There was an uneven distribution of mineral element concentrations in wheat grain. Shorts and bran fractions had the highest mineral element concentrations, whereas flours from break and reduction had low concentrations. Compared with those in the central endosperm, the concentration of inorganic phosphorus (Pi) decreased the most with decreasing flour yield, whereas the concentration of phytic acid P (PAP), phytase activity, and Ca decreased the least. Pi was the most concentrated element in the aleurone, whereas PAP, phytase activity, and Ca were the least, compared to those in the central endosperm. Milling technique through adjusting flour yield can be used to improve the element composition of flour.     

Mineral element distributions in milling fractions of Chinese wheats

Malnutrition related to micronutrient deficiency can create immense economic and societal problems. The objective of this study was to quantify the mineral element concentration distribution in milled fractions, using 43 common wheat (Triticum aestivum L.) cultivars sown in Jinan, China during the 2005–2006 crop season. All 43 cultivars had low Fe (average 28.2 mg Kg⁻¹) and Zn (28.6 mg Kg⁻¹) concentrations, and wide ranges of variation for mineral element concentrations. Highly significant effects among milling fractions and cultivars on all traits were observed, with fraction effect being the larger. There was an uneven distribution of mineral element concentrations in wheat grain. Shorts and bran fractions had the highest mineral element concentrations, whereas flours from break and reduction had low concentrations. Compared with those in the central endosperm, the concentration of inorganic phosphorus (Pi) decreased the most with decreasing flour yield, whereas the concentration of phytic acid P (PAP), phytase activity, and Ca decreased the least. Pi was the most concentrated element in the aleurone, whereas PAP, phytase activity, and Ca were the least, compared to those in the central endosperm. Milling technique through adjusting flour yield can be used to improve the element composition of flour.     

Genetics and chemistry of pigments in wheat grain – A review

Cereal grains contain many phytochemicals, some of which significantly influence the grain colour. Anthocyanins are accumulated in the aleurone or pericarp layer and give blue, purple or combination of these colours. Flavonoids, such as yellow C-glycosides of flavones, flavonols, flavanonols, proanthocyanidins and reddish-coloured phlobaphenes are mainly present in the outer layer of grains while carotenoids that are responsible for yellow grain are in the endosperm. Therefore, accumulation of these pigments in the grain can represent an important target in breeding programmes aimed at increasing the concentrations of bioactive components in grain and products. This review therefore summarises our current knowledge of anthocyanin and carotenoid pigments, their genetic control and variation in levels in different wheat lines.     

小麦籽粒发育时期Puroindolines蛋白与硬度的关系

为探讨Puroindolines蛋白的表达特点与籽粒硬度的关系,采用改进的SDS-PAGE凝胶分析了不同硬度小麦品种的籽粒在各个发育时期Puroindolines蛋白的表达.结果表明,不同硬度的小麦籽粒中总Puroindolines(PinA和PinB)蛋白的表达量差异不大,但与胚乳淀粉颗粒结合的Puroindolines蛋白量差异非常明显:在籽粒发育的不同时期,软质小麦籽粒淀粉粒表面结合的Puroindolines蛋白量显著高于硬质小麦;基因型同为野生型但硬度有差异的品种,籽粒较软的材料其淀粉粒表面结合的Puroindolines蛋白量也明显高于较硬的材料,说明该蛋白的结合特性是决定籽粒硬度的直接原因.结果还表明,胚乳中水溶性戊聚糖与籽粒硬度关系密切.     

Grain color development and the inheritance of high anthocyanin blue aleurone and purple pericarp in spring wheat (Triticum aestivum L.)

There is renewed interest in breeding for high anthocyanin content in wheat due to its antioxidant potential. A series of adapted spring wheat lines were developed with blue aleurone or purple pericarp. The development of anthocyanin concentration and color of these selected lines was measured during grain filling for two field seasons at Saskatoon, Canada. In addition, the inheritance of the blue aleurone and purple pericarp was studied. Anthocyanin concentration increased rapidly during grain development and then decreased before maturity. Anthocyanin concentration was highest in PIG03008, a purple pericarp wheat. For mature grain, genotypic variation for anthocyanin concentration was statistically significant while the year and genotype by year interaction were not, facilitating the breeding progress. Blue aleurone was shown to be controlled by a single dominant gene in BC populations whereas purple pericarp appeared to be controlled by two loci with a segregation ratio of 11 purple: 5 white in F₂ populations. The results indicate that breeding high anthocyanin blue or purple wheat is feasible.     

Isolation of the wheat aleurone layer for 2D electrophoresis and proteomics analysis

Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the aleurone layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral layers including the aleurone layer or only the aleurone layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the aleurone layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat.     

Changes in common wheat grain milling behavior and tissue mechanical properties following ozone treatment

Ozone treatment (10 g/kg) of common wheat grains with a new patented process, Oxygreen^®, used before milling was found to significantly reduce (by 10–20%) the required energy at breaking stage whatever the grain hardness and without changes in the flour yield. Detailed study of each of the milling steps undertaken on a hard type cultivar showed that both the breaking and the reduction energy were decreased. Reduction of the coarse bran yield was also observed concomitantly with an increase in the yield of white shorts. Biochemical characterization of the milling fractions pointed out changes in technological flour properties as starch damage reduction, aleurone content enrichment and increase of insoluble glutenin polymers. Measurement of wheat grain tissue mechanical properties showed that ozone treatment leads to reduction of the aleurone layer extensibility and affects the local endosperm resistance to rupture. These data as well as the direct effect of ozone oxidation on biochemical compounds could explain the observed changes in milling energy, bran and shorts yield and flour composition.     

The Endosperm Morphology of Rice and its Wild Relatives as Observed by Scanning Electron Microscopy

While cultivated rice, Oryza sativa, is arguably the world’s most important cereal crop, there is little comparative morphological information available for the grain of rice wild relatives. In this study, the endosperm of 16 rice wild relatives were compared to O. sativa subspecies indica and O. sativa subspecies japonica using scanning electron microscopy. Although the aleurone, starch granules, protein bodies and endosperm cell shapes of the cultivated and non-cultivated species were similar, several differences were observed. The starch granules of some wild species had internal channels that have not been reported in cultivated rice. Oryza longiglumis, Microlaena stipoides and Potamophila parviflora, had an aleurone that was only one-cell thick in contrast to the multiple cell layers observed in the aleurone of the remaining Oryza species. The similarity of the endosperm morphology of undomesticated species with cultivated rice suggests that some wild species may have similar functional properties. Obtaining a better understanding of the wild rice species grain ultrastructure will assist in identifying potential opportunities for development of these wild species as new cultivated crops or for their inclusion in plant improvement programmes.     

水稻胚乳细胞发育的结构观察及其矿质元素分析

 为了探明水稻胚乳发育的过程和糊粉层形成的机理, 用光学显微镜和电子显微镜观察了水稻糊粉层细胞和内胚乳细胞在颖果发育过程中的结构变化,用能谱仪分析了胚乳细胞中元素的种类和相对含量。结果表明, 糊粉层细胞是由胚乳表层细胞转化而来的。糊粉层细胞中的P、K、Mg和Ca等矿质元素含量要明显高于内胚乳细胞。发育初期糊粉层细胞中富含线粒体、圆球体和小液泡;发育中后期小液泡积累蛋白质和矿质元素而形成糊粉粒。在发育中后期,内胚乳细胞随着细胞内淀粉体的充实,细胞核发生形变而衰亡;而糊粉层细胞的核在发育过程中不消亡。糊粉层的形成与表层细胞积聚矿质和脂类等“灌浆废物”(指非内胚乳细胞的贮藏物)有关。因而,转运灌浆物质多的胚乳背部,其糊粉层细胞的层数要比腹部和侧部多。谷物胚乳发育分为游离核期、细胞化期、分化期和成熟期四个时期。     

Puroindoline genes introduced into durum wheat reduce milling energy and change milling behavior similar to soft common wheats

Grain physical characteristics and milling behavior of a durum wheat line in which both wild-type puroindoline genes were translocated and stabilized after backcrossing (Svevo-Pin) were compared with the parent line (Svevo). The only observed differences between grain characteristics were the mechanical resistance and starchy endosperm porosity revealed through vitreosity measurement. A significant increase of flour and a decrease of semolina yield and break milling energy were observed from Svevo-Pin in comparison with the non-recombinant parent line in accordance to the lower grain mechanical resistance and higher porosity measurements. Moreover, the particle size distribution shown for Svevo-Pin flour appeared consistent with a lower adhesion between starch granules and the protein matrix attributed to the presence of wild-type puroindolines. Coarse bran yield was conversely increased. This appeared to be due to a lower starchy endosperm recovery as a higher proportion of grain starch was found in this bran fraction. Flour from the durum parent line was inversely enriched in phytic acid, a cellular marker of the aleurone layer. Starch damage was also lower in Svevo-Pin flours in comparison with Svevo. All of the observed differences between translocation and parent lines were confirmed independent of the culture growth conditions (n = 12).