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The grains of cereal are able to synthesize and store various protein inhibitors of proteases and amylases. Despite some scientific breakthroughs, there is insufficient information about α-amylase/subtilysin protein inhibitor of wheat grains. Furthermore, when baking bread from germinated wheat grains, the excess activity of α-amylase, which contributed to the restoration of its quality, is suppressed by an inhibitor added from the outside. In our work, the localization of the α-amylase protein inhibitor (PI) in the endosperm and peripheral parts of the grain (shell, aleurone) was determined. In the endosperm, the protein may be free or associated with starch granules. No inhibitor detected in the germ of grain. PI inhibits the catalytic center of Amy1α-amylase of wheat grain, having little influence on the center responsible for the binding of the enzyme to starch granules. The activity and composition of α-amylase in nine samples of wheat meal with high autolytic activity were analyzed (the number of falls is from 248 to 72). FN 91 sec grist showed the most heterogeneous spectrum to the effect of mixtures of wheat's inhibitor and raw extract from grain bran that were taken from the previous studies on α-amylase enzyme. The results of the experiment showed that exposure to the addition of two drugs of purified and raw inhibitors to grist extract with a low number of drops led to a decrease in the activity of α-amylase (Amy1). The novelty of the work was found to inhibit the localization of protein inhibitors and the catalytic center of wheat α-amylase. [ABSTRACT FROM AUTHOR]

We have a limited number of landraces and only a few of them have been used in breeding programs. In the Korean peninsula, maize was introduced before the middle of the 16th century and cultivated widely thereafter. Over 3000 Korean maize landraces have been collected and deposited in the Genebank of the National Agrobiodiversity Center, Korea, which is about a half of total maize germplasm stored in the Genebank. The majority of Korean maize accessions have been maintained as local landraces, implying the preservation of diverse morphological traits with a wide range of variation. However, there has been a few studies for discovering phenotypic characteristics of the Korean maize landraces. We collected phenotype data of 2699 maize germplasm which can be divided by geographic origin (Korean or overseas) and breeding progress (landraces or breeding lines). Principal component analysis demonstrated that the phenotypic variations of the collected maize germplasm were largely explained by flowering time, kernel yields, and stalk diameter traits. From the phenotype data analysis, 276 accessions were selected as a core collection. We also investigated endosperm phenotypes for the core collection and found novel multilayer aleurone germplasms. This indicates that Korean landraces have maintained the original genetic variation which has largely been lost during the modern breeding programs. These findings suggest that the core collection could be a valuable resource for understating and exploiting maize germplasm.

Summary: The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain‐filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type‐specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain‐filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub‐aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two‐time points. Cell‐type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell‐type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain‐filling processes. [ABSTRACT FROM AUTHOR]

Main conclusion: Genetic loci, particularly those with an effect in the independent panel, could be utilised to further reduce LMA expression when used with favourable combinations of genes known to affect LMA. Late maturity α-amylase (LMA) is a grain quality defect involving elevated α-amylase within the aleurone of wheat (Triticum aestivum L.) grains. The genes known to affect expression are the reduced height genes Rht-B1 (chromosome 4B) and Rht-D1 (chromosome 4D), and an ent-copalyl diphosphate synthase gene (LMA-1) on chromosome 7B. Other minor effect loci have been reported, but these are poorly characterised and further genetic understanding is needed. In this study, twelve F4-derived populations were created through single seed descent, genotyped and evaluated for LMA. LMA-1 haplotype C and the Rht-D1b allele substantially reduced LMA expression. The alternative dwarfing genes Rht13 and Rht18 had no significant effect on LMA expression. Additional quantitative trait loci (QTL) were mapped at 16 positions in the wheat genome. Effects on LMA expression were detected for four of these QTL in a large independent panel of Australian wheat lines. The QTL detected in mapping populations and confirmed in the large independent panel provide further opportunity for selection against LMA, especially if combined with Rht-D1b and/or favourable haplotypes of LMA-1. [ABSTRACT FROM AUTHOR]

Utilization of grains of local grasses by Australia's First Nations people for food and connection to Country has largely been lost due to colonization. Native Australian grain production has the potential to deliver environmental, economic, nutritional and cultural benefits to First Nations people and the wider community. Revitalization of the native grain food system can only be achieved if relevant properties of the grains are elucidated. This study aimed to characterize the grain structure and histochemistry of four Australian native grasses: Dactyloctenium radulans (Button Grass), Astrebla lappacea (Curly Mitchell Grass), Panicum decompositum (Native Millet) and Microlaena stipoides (Weeping Grass). For these species, as well as wheat and sorghum, whole-grain images were obtained via stereo microscopy, starch and the embryo were visualized, and sections of fixed grains were imaged via bright-field and fluorescence microscopy. The shape, size and colour of the whole native grains varied between the species. The aleurone layer was one-cell thick in the native species, as in the domesticated grains, except for Weeping Grass, which had a two-cell-thick aleurone. In the native grains, endosperm cell walls appeared thinner than in wheat and sorghum. Starch granules in Button Grass, Curly Mitchell Grass and Native Millet were found mainly in the central region of the starchy endosperm, with very few granules in the sub-aleurone layer, whereas Weeping Grass had abundant starch in the sub-aleurone. Protein appeared most abundant in the aleurone and sub-aleurone layers of the native grains, although in Button Grass, the starchy endosperm was observed to be rich in protein, as in wheat and sorghum. As a proportion of the whole grain, the embryo was larger in the native species than in wheat. The differences found in the grain properties among the four native Australian species have important implications for the agri-food industry in a changing climate. [ABSTRACT FROM AUTHOR]

Endosperm development in barley starts with the formation of a multinucleate syncytium, followed by cellularization in the ventral part of the syncytium generating endosperm transfer cells (ETCs) as first differentiating subdomain, whereas aleurone (AL) cells will originate from the periphery of the enclosing syncytium. Positional signaling in the syncytial stage determines cell identity in the cereal endosperm. Here, we performed a morphological analysis and employed laser capture microdissection (LCM)-based RNA-seq of the ETC region and the peripheral syncytium at the onset of cellularization to dissect developmental and regulatory programs directing cell specification in the early endosperm. Transcriptome data revealed domain-specific characteristics and identified two-component signaling (TCS) and hormone activities (auxin, ABA, ethylene) with associated transcription factors (TFs) as the main regulatory links for ETC specification. On the contrary, differential hormone signaling (canonical auxin, gibberellins, cytokinin) and interacting TFs control the duration of the syncytial phase and timing of cellularization of AL initials. Domain-specific expression of candidate genes was validated by in situ hybridization and putative protein–protein interactions were confirmed by split-YFP assays. This is the first transcriptome analysis dissecting syncytial subdomains of cereal seeds and provides an essential framework for initial endosperm differentiation in barley, which is likely also valuable for comparative studies with other cereal crops. [ABSTRACT FROM AUTHOR]

Rice bran oil, a valuable ingredient of rice bran (composed mainly of embryo and aleurone), is gaining increasing attention for its abundance and benefits for human health. To increase production, breeders have selected for enlarged embryos and thickened aleurone layers. However, breeding for the latter is impeded by the time-consuming and labor-intensive process of observation of aleurone traits. Here, we established a new method for visualizing aleurone layers comprising embedding of mature grains in a plate, dichromatic staining of half grains, and computer-assisted image analysis. With this ‘plate method’, a batch of up to 100 grains fixed on the plate can be handled and examined more efficiently than the standard cryomicrotome method, which only processes the grains individually. In addition, the results obtained from the plate method were highly correlated with that of the cryomicrotome method in terms of aleurone area (r = 0.92) and mean aleurone thickness (r = 0.93). This new method allowed us to rapidly assess the aleurone phenotypes of more than 22,000 mutagenized grains of ‘Mizuhochikara’, with 700–1000 grains per day. As a result, one mutant line with thickened aleurone layer was successfully isolated.

Summary: Aleurone represents a fraction of the outer endosperm in wheat kernels composed of a single‐cell layer containing a variety of nutrients, including dietary fibre (mainly arabinoxylan, β‐glucan, and cellulose), proteins, minerals, vitamins, and antioxidants (ferulic acid). In this study, aleurone‐rich fraction (0%–100%) was added to wheat flour to examine the microstructure of the bread and the crystal structure of the starch after the addition using scanning electron microscopy and X‐ray diffraction. The maximum in vitro hydrolysis rate of bread with 10%–100% aleurone‐rich fraction addition ranged from 35.69% to 65.93%, which was significantly lower than that of white bread (68.76%). Low‐field nuclear magnetic resonance revealed an increase in the amount of bound water in bread after the addition of the aleurone‐rich fraction. Further, bread containing the aleurone‐rich fraction had a higher antioxidant capacity, as evaluated by DPPH• and •OH scavenging activity. Therefore, these results indicate that the aleurone‐rich fraction may be a beneficial ingredient for the manufacture of low‐calorie and high‐fibre foods. [ABSTRACT FROM AUTHOR]

SUMMARY: The maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1‐st (stippled) allele with a novel r1 5′ promoter region homologous to 5′ regions from nkd2‐B73. R1‐sc:124 (an R1‐st derivative) shows increased and earlier expression than a standard R1‐g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1‐sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1‐sc:124 resembled nkd2‐W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1‐sc:124. The r1‐sc:m3 allele is derived from R1‐sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis‐splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5′ region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2‐r1 rearrangement may be a recent event. Significance Statement: The iconic maize R1 gene encodes a HLH transcription factor that conditions anthocyanin pigmentation in aleurone, and has been instrumental in studies of gene expression and transposons. The R1‐sc124 allele is foundational to important genome mutagenesis projects and was created by an intrachromosomal rearrangement that brought regulatory sequences from the nkd2 gene, 1 Mb away, to produce elevated and ectopic R1 expression, resulting in ectopic downstream gene regulation in starchy endosperm tissue. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the concentration and distribution of a number of macro- and micronutrients in response to imbibition in habanero pepper seeds extracted from fruits with different stages of maturity and postharvest storage times, as well as in their seedlings. Fruits were harvested unripe, half-ripe, and ripe, and were subjected to storage for 0 (control), 7, and 14 days postharvest prior to seed extraction. An X-ray microfluorescence analysis was carried out on seeds pre- and post-imbibition, as well as on seedlings at 10 and 14 days after sowing. K, Ca, Fe, P, Mg, and Mn were detected by elemental analysis. The results indicated that the elements had a higher concentration and distribution in seeds extracted from half-ripe fruits and ripe unstored fruits, as well as in seeds extracted from fruits stored for 7 and 14 days postharvest. K and Ca were the elements with the highest distributions and concentrations in seeds and seedlings pre- and post-imbibition compared to the other elements. At all maturity stages, postharvest storage increased the concentration and distribution of mineral nutrients in seeds and seedlings before and after imbibition. Storage translocated mineral elements to the radicle before germination, increased meristem growth in emerged seedlings, decreased electrical conductivity, and increased germination. Fourteen days of postharvest storage increased the distribution of macro- and micronutrients in immature seeds, decreasing electrical conductivity, potentiating germination, and improving mineral element distribution in seedlings. [ABSTRACT FROM AUTHOR]

Purpose: Aleurone is a cereal bran fraction containing a variety of beneficial nutrients including polyphenols, fibers, minerals and vitamins. Animal and human studies support the beneficial role of aleurone consumption in reducing cardiovascular disease (CVD) risk. Gut microbiota fiber fermentation, polyphenol metabolism and betaine/choline metabolism may in part contribute to the physiological effects of aleurone. As primary objective, this study evaluated whether wheat aleurone supplemented foods could modify plasma homocysteine. Secondary objectives included changes in CVD biomarkers, fecal microbiota composition and plasma/urine metabolite profiles. Methods: A parallel double-blind, placebo-controlled and randomized trial was carried out in two groups of obese/overweight subjects, matched for age, BMI and gender, consuming foods supplemented with either aleurone (27 g/day) (AL, n = 34) or cellulose (placebo treatment, PL, n = 33) for 4 weeks. Results: No significant changes in plasma homocysteine or other clinical markers were observed with either treatment. Dietary fiber intake increased after AL and PL, animal protein intake increased after PL treatment. We observed a significant increase in fecal Bifidobacterium spp with AL and Lactobacillus spp with both AL and PL, but overall fecal microbiota community structure changed little according to 16S rRNA metataxonomics. Metabolomics implicated microbial metabolism of aleurone polyphenols and revealed distinctive biomarkers of AL treatment, including alkylresorcinol, cinnamic, benzoic and ferulic acids, folic acid, fatty acids, benzoxazinoid and roasted aroma related metabolites. Correlation analysis highlighted bacterial genera potentially linked to urinary compounds derived from aleurone metabolism and clinical parameters. Conclusions: Aleurone has potential to modulate the gut microbial metabolic output and increase fecal bifidobacterial abundance. However, in this study, aleurone did not impact on plasma homocysteine or other CVD biomarkers. Trial Registration: The study was registered at ClinicalTrials.gov (NCT02067026) on the 17th February 2014. [ABSTRACT FROM AUTHOR]

Endosperm development in barley starts with the formation of a multinucleate syncytium, followed by cellularization in the ventral part of the syncytium generating endosperm transfer cells (ETCs) as first differentiating subdomain, whereas aleurone (AL) cells will originate from the periphery of the enclosing syncytium. Positional signaling in the syncytial stage determines cell identity in the cereal endosperm. Here, we performed a morphological analysis and employed laser capture microdissection (LCM)-based RNA-seq of the ETC region and the peripheral syncytium at the onset of cellularization to dissect developmental and regulatory programs directing cell specification in the early endosperm. Transcriptome data revealed domain-specific characteristics and identified two-component signaling (TCS) and hormone activities (auxin, ABA, ethylene) with associated transcription factors (TFs) as the main regulatory links for ETC specification. On the contrary, differential hormone signaling (canonical auxin, gibberellins, cytokinin) and interacting TFs control the duration of the syncytial phase and timing of cellularization of AL initials. Domain-specific expression of candidate genes was validated by in situ hybridization and putative protein–protein interactions were confirmed by split-YFP assays. This is the first transcriptome analysis dissecting syncytial subdomains of cereal seeds and provides an essential framework for initial endosperm differentiation in barley, which is likely also valuable for comparative studies with other cereal crops.

Main conclusion: α-Amylase synthesis by wheat aleurone during grain development (late maturity α-amylase) appears to be independent of gibberellin unlike α-amylase synthesis by aleurone during germination or following treatment with exogenous GA. Late-maturity α-amylase (LMA) in wheat (Triticum aestivum L.) involves the synthesis of α-amylase by the aleurone tissue during grain development. Previous research identified a putative ent-copalyl diphosphate synthase gene, coding for an enzyme that controls the first step in gibberellin biosynthesis, that underlies the major genetic locus involved in variation in LMA phenotype. The reported results for gene transcript analysis, preliminary gibberellin analysis and the effects of DELLA mutants on LMA phenotype appeared to be consistent with involvement of gibberellin but did not provide definitive proof of a causal link. Conversely, several observations do not appear to be consistent with this hypothesis. In this current study, LMA phenotype, gibberellin profiles and ABA content were recorded for experiments involving susceptible and resistant genotypes, gibberellin biosynthesis inhibitors, genetic lines containing different LMA quantitative trait loci and treatment of distal halves of developing grains with exogenous gibberellin. The results suggested that gibberellin may not be a prerequisite for LMA expression and further that the mechanism involved in triggering α-amylase synthesis did not correspond to the model proposed for germination and gibberellin challenged aleurone of ripe grain. The results provide new insight into LMA and highlight the need to investigate alternate pathways for the induction of α-amylase gene transcription, the function of novel 1-β-OH gibberellins and other functions of DELLA proteins in developing grains. [ABSTRACT FROM AUTHOR]

Rice bran oil, a valuable ingredient of rice bran (composed mainly of embryo and aleurone), is gaining increasing attention for its abundance and benefits for human health. To increase production, breeders have selected for enlarged embryos and thickened aleurone layers. However, breeding for the latter is impeded by the time-consuming and labor-intensive process of observation of aleurone traits. Here, we established a new method for visualizing aleurone layers comprising embedding of mature grains in a plate, dichromatic staining of half grains, and computer-assisted image analysis. With this 'plate method', a batch of up to 100 grains fixed on the plate can be handled and examined more efficiently than the standard cryomicrotome method, which only processes the grains individually. In addition, the results obtained from the plate method were highly correlated with that of the cryomicrotome method in terms of aleurone area (r = 0.92) and mean aleurone thickness (r = 0.93). This new method allowed us to rapidly assess the aleurone phenotypes of more than 22,000 mutagenized grains of 'Mizuhochikara', with 700–1000 grains per day. As a result, one mutant line with thickened aleurone layer was successfully isolated. [ABSTRACT FROM AUTHOR]

Protein storage vacuoles (PSVs) in aleurone cells coalesce during germination, and this process is highly coupled with mobilization of PSV reserves, allowing de novo synthesis of various hydrolases in aleurone cells for endosperm degradation. Here we show that in barley (Hordeum vulgare L.) oleosins, the major integral proteins of oleosomes are encoded by four genes (HvOle1 to 4), and the expression of HvOle1 and HvOle3 is strongly up-regulated by abscisic acid (ABA), which shows antagonism to gibberellic acid. In aleurone cells, all HvOLEs were subcellularly targeted to the tonoplast of PSVs. Gain-of-function analyses revealed that HvOLE3 effectively delayed PSV coalescence, whereas HvOLE1 only had a moderate effect, with no notable effect of HvOLE2 and 4. With regard to longevity, HvOLE3 chiefly outperformed other HvOLEs, followed by HvOLE1. Experiments swapping the N- and C-terminal domain between HvOLE3 and other HvOLEs showed that the N-terminal region of HvOLE3 is mainly responsible, with some positive effect by the C-terminal region, for mediating the specific preventive effect of HvOLE3 on PSV coalescence. Three ACGT-core elements and the RY-motif were responsible for ABA induction of HvOle3 promoter activity. Transient expression assays using aleurone protoplasts demonstrated that transcriptional activation of the HvOle3 promoter was mediated by transcription factors HvABI3 and HvABI5, which acted downstream of protein kinase HvPKABA1. [ABSTRACT FROM AUTHOR]

Abstract Background Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. Results An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. Conclusions Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

The major tissues of the cereal endosperm are the starchy endosperm (SE) in the inner and the aleurone layer (AL) at the outer periphery. The fates of the cells that comprise these tissues are determined according to positional information; however, our understanding of the underlying molecular mechanisms remains limited. Here, we conducted a high-resolution spatiotemporal analysis of the rice endosperm transcriptome during early cellularization. In rice, endosperm cellularization proceeds in a concentric pattern from a primary alveolus cell layer, such that developmental progression can be defined by the number of cell layers. Using laser-capture microdissection to obtain precise tissue sections, transcriptomic changes were followed through five histologically defined stages of cellularization from the syncytial to 3-cell layer (3 L) stage. In addition, transcriptomes were compared between the inner and the outermost peripheral cell layers. Large differences in the transcriptomes between stages and between the inner and the peripheral cells were found. SE attributes were expressed at the alveolus-cell-layer stage but were preferentially activated in the inner cell layers that resulted from periclinal division of the alveolus cell layer. Similarly, AL attributes started to be expressed only after the 2 L stage and were localized to the outermost peripheral cell layer. These results indicate that the first periclinal division of the alveolus cell layer is asymmetric at the transcriptome level, and that the cell-fate-specifying positional cues and their perception system are already operating before the first periclinal division. Several genes related to epidermal identity (i.e., type IV homeodomain-leucine zipper genes and wax biosynthetic genes) were also found to be expressed at the syncytial stage, but their expression was localized to the outermost peripheral cell layer from the 2 L stage onward. We believe that our findings significantly enhance our knowledge of the mechanisms underlying cell fate specification in rice endosperm. [ABSTRACT FROM AUTHOR]

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Summary: Understanding the mechanisms of iron trafficking in plants is key to enhancing the nutritional quality of crops. Because it is difficult to image iron in transit, we currently have an incomplete picture of the route(s) of iron translocation in developing seeds and how the tissue‐specific distribution is established.We have used a novel approach, combining iron‐57 (57Fe) isotope labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), to visualize iron translocation between tissues and within cells in immature wheat grain, Triticum aestivum.This enabled us to track the main route of iron transport from maternal tissues to the embryo through the different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, with confirmation provided by histological staining and transmission electron microscopy energy dispersive X‐ray spectroscopy (TEM‐EDS). Almost all iron in both control and transgenic grains was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Furthermore, a new type of iron body, highly enriched in 57Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids.Correlation of the 57Fe enrichment profiles obtained by NanoSIMS with tissue‐specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification strategies. [ABSTRACT FROM AUTHOR]

Summary: Various observations show a lamellar organisation of wheat aleurone cell walls, that likely acted as barriers to regulate the water diffusion rate in wheat grains. In this study, arabinoxylan (AX) and (1,3)(1,4)‐β‐glucan (BG) alternating multilayer films which mimic cell wall of aleurone structure were prepared. Twenty layers of alternating multilayer films of AX/BG were prepared with individual thicknesses of 20 µm. The water diffusion and water mobility of the films were determined using dynamic vapour sorption and time‐domain nuclear magnetic resonance spectroscopy. The AX/BG alternating multilayer films exhibited unique mechanical and hydration properties, which could be explained by their unique nanostructure and microporosity. The edible films with this structure were found to exhibit high ultimate stress and ultimate strain, and low rates of water diffusion. [ABSTRACT FROM AUTHOR]

Mineral malnutrition affects billions of people all over the world and biofortification of staple crops provides a potential way to alleviate dietary mineral deficiencies. For example, nutritional quality is an important breeding target for fresh waxy maize (Zea mays L.), which is widely consumed in Asian countries. Successful improvement of mineral composition will require comprehensive profiling of the mineral composition of maize varieties and an understanding of the capacity for maize grains to accumulate minerals. Here, using inductively coupled plasma absorption emission spectrometry, we quantified 12 minerals from the seeds of 47 maize varieties, including 25 Korean landraces. We also compared the mineral contents in varieties with different seed starch profiles: waxy maize (which contains 100% amylopectin), dent maize (roughly 75% amylopectin and 25% amylose), and flint maize (similar to dent maize). The amounts of potassium, phosphorus, and sulfur were correlated with seed texture, waxy maize having higher amounts of phosphorus and potassium than dent maize and lower amounts of sulfur than flint maize or dent maize. In addition, a positive relationship was detected between the amount of phosphorus and that of potassium, magnesium, and manganese. These results provide information on maize seed mineral composition and indicate that it could be affected by starch composition. Furthermore, the landraces that exhibit high mineral contents could be used as germplasm materials for breeding programs aimed at producing biofortified maize cultivars.

The aim of this study was to evaluate the concentration and distribution of a number of macro- and micronutrients in response to imbibition in habanero pepper seeds extracted from fruits with different stages of maturity and postharvest storage times, as well as in their seedlings. Fruits were harvested unripe, half-ripe, and ripe, and were subjected to storage for 0 (control), 7, and 14 days postharvest prior to seed extraction. An X-ray microfluorescence analysis was carried out on seeds pre- and post-imbibition, as well as on seedlings at 10 and 14 days after sowing. K, Ca, Fe, P, Mg, and Mn were detected by elemental analysis. The results indicated that the elements had a higher concentration and distribution in seeds extracted from half-ripe fruits and ripe unstored fruits, as well as in seeds extracted from fruits stored for 7 and 14 days postharvest. K and Ca were the elements with the highest distributions and concentrations in seeds and seedlings pre- and post-imbibition compared to the other elements. At all maturity stages, postharvest storage increased the concentration and distribution of mineral nutrients in seeds and seedlings before and after imbibition. Storage translocated mineral elements to the radicle before germination, increased meristem growth in emerged seedlings, decreased electrical conductivity, and increased germination. Fourteen days of postharvest storage increased the distribution of macro- and micronutrients in immature seeds, decreasing electrical conductivity, potentiating germination, and improving mineral element distribution in seedlings.

Minerals are stored in the aleurone layer and embryo during maize seed development, but how they affect endosperm development and activity is unclear. Here, we cloned the gene underlying the classic maize kernel mutant shrunken4 (sh4) and found that it encodes the YELLOW STRIPE-LIKE oligopeptide metal transporter ZmYSL2. sh4 kernels had a shrunken phenotype with developmental defects in the aleurone layer and starchy endosperm cells. ZmYSL2 showed iron and zinc transporter activity in Xenopus laevis oocytes. Analysis using a specific antibody indicated that ZmYSL2 predominately accumulated in the aleurone and sub-aleurone layers in endosperm and the scutellum in embryos. Specific iron deposition was observed in the aleurone layer in wild-type kernels. In sh4, however, the outermost monolayer of endosperm cells failed to accumulate iron and lost aleurone cell characteristics, indicating that proper functioning of ZmYSL2 and iron accumulation are essential for aleurone cell development. Transcriptome analysis of sh4 endosperm revealed that loss of ZmYSL2 function affects the expression of genes involved in starch synthesis and degradation processes, which is consistent with the delayed development and premature degradation of starch grains in sh4 kernels. Therefore, ZmYSL2 is critical for aleurone cell development and starchy endosperm cell activity during maize seed development. [ABSTRACT FROM AUTHOR]

Starch is synthesized in the endosperm of developing barley grain, where it functions as the primary source of stored carbohydrate. In germinated grain these starch reserves are hydrolyzed to small oligosaccharides and glucose, which are transported to the embryo to support the growth of the developing seedling. Some of the mobilized glucose is transiently stored as starch in the scutellum of germinated grain. These processes are crucial for early seedling vigor, which is a key determinant of crop productivity and global food security. Several starch synthases (SS), starch-branching enzymes (SBEs), and starch debranching enzymes (isoamylases, ISA), together with a limit dextrinase (LD), have been implicated in starch synthesis from nucleotide-sugar precursors. Starch synthesis occurs both in the developing endosperm and in the scutellum of germinated grain. For the complete depolymerization of starch to glucose, α-amylase (Amy), β-amylase (Bmy), isoamylase (ISA), limit dextrinase (LD), and α-glucosidase (AGL) are required. Most of these enzymes are encoded by gene families of up to 10 or more members. Here RNA-seq transcription data from isolated tissues of intact developing and germinated barley grain have allowed us to identify the most important, specific gene family members for each of these processes in vivo and, at the same time, we have defined in detail the spatio-temporal coordination of gene expression in different tissues of the grain. A transcript dataset for 81,280 genes is publicly available as a resource for investigations into other cellular and biochemical processes that occur in the developing grain from 6 days after pollination.

BACKGROUND Cereal co‐products rich in dietary fibres are increasingly used in animal feed. The high fibre content decreases the digestibility and reduces the nutrient and energy availability, resulting in lower nutritive value. Therefore, this study investigated the ability of two carbohydrase complexes to solubilize cell‐wall polysaccharides, in particular arabinoxylan (AX), from different cereal fractions of wheat, maize, and rice using an in vitro digestion model of the pig gastric and small intestinal digestive system. The first complex (NSPase 1) was rich in cell‐wall‐degrading enzymes, whereas the second complex (NSPase 2) was additionally enriched with xylanases and arabinofuranosidases. The extent of solubilization of insoluble cell‐wall polysaccharides after in vitro digestion was evaluated with gas–liquid chromatography and an enzymatic fingerprint of the AX oligosaccharides was obtained with high‐performance anion‐exchange chromatography with pulsed amperometric detection. RESULTS: The addition of carbohydrase increased the digestibility of dry matter and solubilized AX in particular, with the greatest effect in wheat fractions and less effect in maize and rice fractions. The solubilization of AX (expressed as xylose release) ranged from 6% to 41%, and there was an increased effect when enriching with xylanases and arabinofuranosidases in wheat aleurone and bran of 19% and 14% respectively. The enzymatic fingerprint of AX oligosaccharides revealed several non‐final hydrolysis products of the enzymes applied, indicating that the hydrolysis of AX was not completed during in vitro digestion. CONCLUSION: These results indicate that the addition of a carbohydrase complex can introduce structural alterations under in vitro digestion conditions, and that enrichment with additional xylanases and arabinofuranosidases can boost this effect in wheat, maize, and rice. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Starch is synthesized in the endosperm of developing barley grain, where it functions as the primary source of stored carbohydrate. In germinated grain these starch reserves are hydrolyzed to small oligosaccharides and glucose, which are transported to the embryo to support the growth of the developing seedling. Some of the mobilized glucose is transiently stored as starch in the scutellum of germinated grain. These processes are crucial for early seedling vigor, which is a key determinant of crop productivity and global food security. Several starch synthases (SS), starch-branching enzymes (SBEs), and starch debranching enzymes (isoamylases, ISA), together with a limit dextrinase (LD), have been implicated in starch synthesis from nucleotide-sugar precursors. Starch synthesis occurs both in the developing endosperm and in the scutellum of germinated grain. For the complete depolymerization of starch to glucose, α-amylase (Amy), β-amylase (Bmy), isoamylase (ISA), limit dextrinase (LD), and α-glucosidase (AGL) are required. Most of these enzymes are encoded by gene families of up to 10 or more members. Here RNA-seq transcription data from isolated tissues of intact developing and germinated barley grain have allowed us to identify the most important, specific gene family members for each of these processes in vivo and, at the same time, we have defined in detail the spatio-temporal coordination of gene expression in different tissues of the grain. A transcript dataset for 81,280 genes is publicly available as a resource for investigations into other cellular and biochemical processes that occur in the developing grain from 6 days after pollination. [ABSTRACT FROM AUTHOR]

Peroxidases (PODs) have many biological functions during the plant life cycle. In maize kernels, endosperm PODs have been identified as biochemical contributors to resistance against Sitophilus zeamais, but their identities have not been determined. In this study, we identified these PODs and determined whether their contributions are basal or inducible. Semi-purification and LC-MS/MS analyses showed that the protein ZmPrx35 is the predominant soluble endosperm POD from kernels of Pob84-C3R. Subsequent time-course analyses after mechanical damage showed that POD activity was regulated in a fluctuating kinetics pattern and that zmprx35 mRNA expression levels reflected this pattern. After 48 h of infestation with S. zeamais or Prostephanus truncatus, soluble endosperm POD activities were 1.38- or 0.85-fold, respectively. Under the same conditions, zmprx35 expression was induced 1.61-fold (S. zeamais infestation) and 1.17-fold (P. truncatus infestation). These findings suggest that ZmPrx35 contributes to the protective responses of aleurone cells against wounding and pest attacks, which could be enhanced/repressed by insect factors. Our data also provide evidence that the mechanisms of resistance of maize Pob84-C3R kernels toward the insect pests S. zeamais and P. truncatus are independent.

Abstract Background Soybean is a globally important oil seed crop. Both the high protein and oil content of soybean seeds make this crop a lucrative commodity. As in higher eukaryotic species with available genomes, the functional annotation of most of soybean’s genes still remains to be investigated. A major hurdle in the functional genomics of soybean is a rapid method to test gene constructs before embarking on stable transformation experiments. Results In this paper we describe the morphology and composition of the persistent single-cell aleurone layer that derives from the endosperm of developing soybean seeds. Its composition compared to cotyledonary tissue indicates the aleurone layer plays a role in both abiotic and biotic stress. The potential utility as the aleurone layer as a transient expression system in soybean was shown. As a near transparent single-cell layer it can be used as a transient expression system to study transgene expression and inter- and intra-cellular targeting as it is amenable to microscopic techniques. Conclusion The transparent single cell aleurone layer was shown to be compositionally comparable to cotyledonary tissue in soybean with an enrichment in oxidative response proteins and shown to be a potential transient expression platform.

Wheat grain from 12 different regions in China was used to study variations in the cell wall structure and chemical composition based on geography. The mobility and migration rate of water in wheat grain during moisture absorption and drying were determined under different relative humidity conditions. Depending on the geography, variations were noted in the thickness and component content of the wheat grain cell wall. Cell wall thickness was positively correlated with the total arabinoxylan (TAX) content. Cell wall thickness and TAX content of the aleurone layer were positively correlated with altitude and negatively correlated with longitude. The water migration rate decreased with the increase of cell wall thickness and TAX content. Nuclear magnetic resonance (NMR) results revealed that grains with thick aleurone cell wall showed increased molecular mobility of water. These findings lay the foundation for further study of water regulation in wheat cell wall. Past studies on the mechanisms of wheat grains formation have focussed on the regulation of related synthase activity and expression pattern of coding genes, but ignored the influence of cell wall microenvironment. Our study indicates that variations in wheat grain cell wall structure depend on the geography, and grains with thick cell walls show improved water retention and molecular mobility. This finding will help guide further studies on water regulation in wheat cell walls. [ABSTRACT FROM AUTHOR]

Peroxidases (PODs) have many biological functions during the plant life cycle. In maize kernels, endosperm PODs have been identified as biochemical contributors to resistance against Sitophilus zeamais , but their identities have not been determined. In this study, we identified these PODs and determined whether their contributions are basal or inducible. Semi-purification and LC-MS/MS analyses showed that the protein ZmPrx35 is the predominant soluble endosperm POD from kernels of Pob84-C3R. Subsequent time-course analyses after mechanical damage showed that POD activity was regulated in a fluctuating kinetics pattern and that zmprx35 mRNA expression levels reflected this pattern. After 48 h of infestation with S. zeamais or Prostephanus truncatus , soluble endosperm POD activities were 1.38- or 0.85-fold, respectively. Under the same conditions, zmprx35 expression was induced 1.61-fold (S. zeamais infestation) and 1.17-fold (P. truncatus infestation). These findings suggest that ZmPrx35 contributes to the protective responses of aleurone cells against wounding and pest attacks, which could be enhanced/repressed by insect factors. Our data also provide evidence that the mechanisms of resistance of maize Pob84-C3R kernels toward the insect pests S. zeamais and P. truncatus are independent. [ABSTRACT FROM AUTHOR]

Background: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. Results: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. Conclusions: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil. [ABSTRACT FROM AUTHOR]

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain. [ABSTRACT FROM AUTHOR]

Wheat bran fibers are considered beneficial to human health through their impact on gut microbiota composition and activity. Here, we assessed the prebiotic potential of selected bran fractions by performing a series of fecal slurry anaerobic fermentation experiments using aleurone as well as total, ultrafine, and soluble wheat bran (swb) as carbon sources. By combining amplicon-based community profiling with a fluorescent in situ hybridization (FISH) approach, we found that incubation conditions favor the growth of Proteobacteria such as Escherichia and Bilophila. These effects were countered in all but one [total wheat bran (twb)] fermentation experiments. Growth of Bifidobacterium species was stimulated after fermentation using ultrafine, soluble, and twb, in the latter two as part of a general increase in bacterial load. Both ultrafine and swb fermentation resulted in a trade-off between Bifidobacterium and Bilophila, as previously observed in human dietary supplementation studies looking at the effect of inulin-type fructans on the human gut microbiota. Aleurone selectively stimulated growth of Dorea and butyrate-producing Roseburia. All fermentation experiments induced enhanced gas production; increased butyrate concentrations were only observed following soluble bran incubation. Our results open perspectives for the development of aleurone as a complementary prebiotic selectively targeting colon butyrate producers.

Background: Soybean is a globally important oil seed crop. Both the high protein and oil content of soybean seeds make this crop a lucrative commodity. As in higher eukaryotic species with available genomes, the functional annotation of most of soybean’s genes still remains to be investigated. A major hurdle in the functional genomics of soybean is a rapid method to test gene constructs before embarking on stable transformation experiments. Results: In this paper we describe the morphology and composition of the persistent single-cell aleurone layer that derives from the endosperm of developing soybean seeds. Its composition compared to cotyledonary tissue indicates the aleurone layer plays a role in both abiotic and biotic stress. The potential utility as the aleurone layer as a transient expression system in soybean was shown. As a near transparent single-cell layer it can be used as a transient expression system to study transgene expression and inter- and intra-cellular targeting as it is amenable to microscopic techniques. Conclusion: The transparent single cell aleurone layer was shown to be compositionally comparable to cotyledonary tissue in soybean with an enrichment in oxidative response proteins and shown to be a potential transient expression platform. [ABSTRACT FROM AUTHOR]

Abstract: Inadequate intake of essential minerals such as iron and zinc is a public health concern in the UK, particularly for girls and young women. Approximately 30% and 50% of the zinc and iron, respectively, in the UK diet is provided by cereals. In wheat, most of the iron and zinc is contained within the aleurone cell layer; however, aleurone is removed during processing of wheat into white flour. While elemental iron powder is added back into white flour at the milling stage, there is no restoration of zinc. Elemental iron powder has very low bioavailability, and therefore, in our current Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club‐funded project, we are investigating the potential use of aleurone as a bioavailable source of minerals that could be added to wheat‐based foods. This work has relevance for the food industry and may establish the use of aleurone as a functional food ingredient for fortification of a range of cereal‐based food products. [ABSTRACT FROM AUTHOR]

Wheat bran fibers are considered beneficial to human health through their impact on gut microbiota composition and activity. Here, we assessed the prebiotic potential of selected bran fractions by performing a series of fecal slurry anaerobic fermentation experiments using aleurone as well as total, ultrafine, and soluble wheat bran (swb) as carbon sources. By combining amplicon-based community profiling with a fluorescent in situ hybridization (FISH) approach, we found that incubation conditions favor the growth of Proteobacteria such as Escherichia and Bilophila. These effects were countered in all but one [total wheat bran (twb)] fermentation experiments. Growth of Bifidobacterium species was stimulated after fermentation using ultrafine, soluble, and twb, in the latter two as part of a general increase in bacterial load. Both ultrafine and swb fermentation resulted in a trade-off between Bifidobacterium and Bilophila, as previously observed in human dietary supplementation studies looking at the effect of inulin-type fructans on the human gut microbiota. Aleurone selectively stimulated growth of Dorea and butyrateproducing Roseburia. All fermentation experiments induced enhanced gas production; increased butyrate concentrations were only observed following soluble bran incubation. Our results open perspectives for the development of aleurone as a complementary prebiotic selectively targeting colon butyrate producers. [ABSTRACT FROM AUTHOR]

Imprinted gene expression in flowering plants predominantly occurs in the triploid endosperm of developing seed. However, endosperm is composed of distinct tissue types. For example, the maize ( Zea mays) endosperm is constituted by two major tissues, starchy endosperm and aleurone. Previous studies in imprinted gene expression have generally assumed that the different tissues constituting endosperm would behavior the same, and hence have not examined them separately. Here, to examine parental-specific expression of imprinted genes in different parts of the seed, eight previously reported maize protein-coding imprinted genes were selected, and analyzed by cleaved amplified polymorphic sequence (CAPS) coupled with Sanger sequencing for transcripts from the various seed tissues collected at 18 days after pollination (DAP). The studied tissues included seed coat, embryo, starchy endosperm and aleurone, which were collected from a pair of reciprocal F1 hybrids produced by crossing inbred lines B73 and Mo17. Six of these eight analyzed imprinted genes showed the same imprinted expression pattern between the starchy endosperm and aleurone, but two showed imprinted expression only in the starchy endosperm. Comparison of the expression pattern of 20 selected imprinted genes in multiple seed tissues and vegetative tissues indicated that the majority (~ 75%) of these imprinted genes exhibited seed-specific or endosperm-specific expression. Our results also uncovered that imprinted genes have a high propensity to be alternatively spliced via intron retention in the developing embryo compared with the other tissues. [ABSTRACT FROM AUTHOR]

The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs) in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.