A plasma membrane-localized transporter remobilizes aleurone layer magnesium for seed germination in rice.

The aleurone layer in cereal grains acts as a major reservoir of essential mineral nutrients, significantly influencing seed germination. However, the molecular mechanism underlying the redistribution of nutrients from the aleurone layer in the germinating seed is still not well understood. Here, in rice, we identified a plasma membrane (PM) localized magnesium transporter, MAGNESIUM RELEASE TRANSPORTER 3 (MGR3), is critical for seed germination. OsMGR3 is predominantly expressed in the aleurone layer cells of endosperm, facilitating magnesium remobilization during germination. Non-invasive Micro-test Technology assay data demonstrated that the loss-of-function of OsMGR3 restrained magnesium efflux from the aleurone layer. In the embryo/endosperm grafting experiment, we observed that the mutation of OsMGR3 in the aleurone layer suppressed the growth and differentiation of the embryo during germination. Furthermore, magnesium fluorescence imaging revealed the osmgr3 mutant seeds showed impaired exportation of aleurone layer-stored magnesium to the embryo, consequently delaying germination. Importantly, we discovered that disrupting OsMGR3 could inhibit pre-harvest sprouting without affecting rice yield and quality. Therefore, the magnesium efflux transporter OsMGR3 in the aleurone layer represents a promising genetic target for future agronomic trait improvement.

HvGST plays a key role in anthocyanin accumulation in colored barley.

Blue aleurone of barley is caused by the accumulation of delphinidin-based derivatives. Although these compounds are ideal nutrients for human health, they are undesirable contaminants in malt brewing. Therefore, the ability to add and remove this trait easily would facilitate breeding barley for different purposes. Here we identified a glutathione S-transferase gene (HvGST) that was responsible for the blue aleurone trait in Tibetan qingke barley by performing a genome-wide association study and RNA-sequencing analysis. Gene variation and expression analysis indicated that HvGST also participates in the transport and accumulation of anthocyanin in purple barley. Haplotype and the geographic distribution analyses of HvGST alleles revealed two independent natural variants responsible for the emergence of white aleurone: a 203-bp deletion causing premature termination of translation in qingke barley and two key single nucleotide polymorphisms in the promoter resulting in low transcription in Western barley. This study contributes to a better understanding of mechanisms of colored barley formation, and provides a comprehensive reference for marker-assisted barley breeding.

Genetic analysis of late-maturity α-amylase in twelve wheat populations.

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.

Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endosperm.

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.

Wheat bran arabinoxylans: Chemical structure, extraction, properties, health benefits, and uses in foods.

Wheat bran (WB) is a well-known and valuable source of dietary fiber. Arabinoxylan (AX) is the primary hemicellulose in WB and can be isolated and used as a functional component in various food products. Typically, AX is extracted from the whole WB using different processes after mechanical treatments. However, WB is composed of different layers, namely, the aleurone layer, pericarp, testa, and hyaline layer. The distribution, structure, and extractability of AX vary within these layers. Modern fractionation technologies, such as debranning and electrostatic separation, can separate the different layers of WB, making it possible to extract AX from each layer separately. Therefore, AX in WB shows potential for broader applications if it can be extracted from the different layers separately. In this review, the distribution and chemical structures of AX in WB layers are first discussed followed by extraction, physicochemical properties, and health benefits of isolated AX from WB. Additionally, the utilization of AX isolated from WB in foods, including cereal foods, packaging film, and the delivery of food ingredients, is reviewed. Future perspectives on challenges and opportunities in the research field of AX isolated from WB are highlighted.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

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.

Wheat bran layers: composition, structure, fractionation, and potential uses in foods.

Wheat bran, the main by-product of dry milling of wheat, is currently mainly used in the animal feed industry, but has attracted attention as a food ingredient owing to its high dietary fiber and phytochemical contents, providing excellent physiological effects. The bran layers (aleurone layer, outer pericarp and intermediate layer) contain different compositions, structures, and nutrients, and have different properties. Each layer, when separated and isolated, potentially could find more extensive applications in foods. This triggered interest in isolating the bran layers using milling and wet- or dry-fractionation techniques based on their chemical or physical properties. The recent progress has allowed the production of commercial products from wheat bran layers, particularly aleurone-rich products, enhancing the value of wheat bran layers and their applications in food. The present review highlights the recent advances in studying the chemical composition including distribution of chemical components, physical structure, biopolymer matrix, and physicochemical properties of each wheat bran layer. Technologies to fractionate wheat bran layers and utilization of different bran layers in foods are discussed and reviewed, providing new strategies for improving the value of wheat bran and utilization of wheat bran in foods.

Barley metallothionein isoforms, MT2b2 and MT4, differentially respond to photohormones in barley aleurone layer and their recombinant forms show different affinity for binding to zinc and cadmium.

Metallothioneins (MTs) are metal-binding proteins that have important roles in the homeostasis of heavy metals. In this study, the two MT genes was studied in response to phytohormones using the barley aleurone layer as a kind of model system. The aleurone layer was isolated from barley embryo-less half grains and was incubated for 24 h with different phytohormones. Based on the results the genes encoding HvMT2b2 and HvMT4 were down-regulated through gibberellic acid (GA), while they were and up-regulated through salicylic acid (SA). Despite this, these two genes were differentially expressed to other hormones. Furthermore, the proteins HvMT2b2 and HvMT4 were heterologous expressed as GST-fusion proteins in E. coli. The HvMT4 and HvMT2b2 heterologous expression in E. coli gives rise to 10- and 3-fold improvements in the accumulation capacity for Zn2+, respectively. Whereas the transgenic E. coli strain that expresses HvMT2b2 could accumulate Cd2+ three-fold higher than control. The expression of HvMT4 did not affect the accumulation of Cd2+. HvMT4 which is known as seed-specific isoform seems to be able to bind to Zn2+ with good affinity and cannot bind Cd2+. In comparison, HvMT2b2 was able to bind both Zn2+ and Cd2+. Therefore HvMT4 could serve a noteworthy role in zinc storage in barley seeds. The expression of HvMT4 is induced by SA 30-fold, concerning the untreated aleurone layer. Such results could provide good insights for the assessment of the effects of phytohormones in the molecular mechanism involved in essential metal storage in cereal seeds.

Laser microdissection transcriptome data derived gene regulatory networks of developing rice endosperm revealed tissue- and stage-specific regulators modulating starch metabolism.

KEY MESSAGE: Laser microdissection applied on the developing rice endosperm revealed tissue- and stage-specific regulators modulating programmed cell death and desiccation tolerance mechanisms in the central starchy endosperm following starch metabolism. Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44 K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Gibberellins in developing wheat grains and their relationship to late maturity α-amylase (LMA).

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-ß-OH gibberellins and other functions of DELLA proteins in developing grains.

Abscisic acid activation of oleosin gene HvOle3 expression prevents the coalescence of protein storage vacuoles in barley aleurone cells.

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.

Impact of wheat aleurone on biomarkers of cardiovascular disease, gut microbiota and metabolites in adults with high body mass index: a double-blind, placebo-controlled, randomized clinical trial.

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.

A wheat aleurone-rich diet improves oxidative stress but does not influence glucose metabolism in overweight/obese individuals: Results from a randomized controlled trial.

BACKGROUND AND AIMS: Aleurone is the innermost layer of wheat bran, rich in fiber, minerals, vitamins, phenolic compounds, and betaine. The metabolic effects of aleurone rich foods are still unknown. Our aim was to investigate the effects of consuming a Wheat Aleurone rich diet vs. a Refined Wheat diet for 8 weeks on fasting and postprandial glycemic and lipid metabolism, inflammation, and oxidative stress in overweight/obese individuals. METHODS AND RESULTS: According to a randomized cross-over study design, 23 overweight/obese individuals, age 56 ± 9 years (M±SD), were assigned to two isoenergetic diet - Wheat Aleurone and Refined Wheat diets - for 8 weeks. The diets were similar for macronutrient composition but different for the aleurone content (40-50 g/day in the Wheat Aleurone diet). After each diet, fasting and postprandial plasma metabolic profile, ferulic acid metabolites and 8-isoprostane concentrations in 24-h urine samples were evaluated. Compared with the Refined Wheat Diet, the Wheat Aleurone Diet increased fasting plasma concentrations of betaine by 15% (p = 0.042) and decreased the excretion of 8-isoprostane by 33% (p = 0.035). Conversely, it did not affect the fasting and postprandial glucose, insulin and triglyceride responses, homocysteine, and C-Reactive Protein concentrations, nor excretion of phenolic metabolites. CONCLUSION: An 8-week Wheat Aleurone Diet improves the oxidative stress and increases plasma betaine levels in overweight/obese individuals with an increased cardiometabolic risk. However, further studies with longer duration and larger sample size are needed to evaluate the benefits of aleurone-rich foods on glucose and lipid metabolism in individuals with more severe metabolic abnormalities. CLINICAL TRIAL REGISTRY NUMBER: NCT02150356, (https://clinicaltrials.gov).

Uncovering the evolutionary origin of blue anthocyanins in cereal grains.

Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only Hordeum vulgare (barley), Triticum aestivum (wheat) and Secale cereale (rye) accumulate delphinidin-derived (blue) anthocyanins in the aleurone layer of grains, whereas Oryza sativa (rice), Zea mays (maize) and Sorghum bicolor (sorghum) do not. The underlying genetic basis for this natural occurrence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleurone to an approximately 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue-coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co-evolved together. We also identified a Pooideae-specific flavonoid 3',5'-hydroxylase (F3'5'H) lineage, termed here Mo_F35H2, which has a higher amino acid similarity with eudicot F3'5'Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 that underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, through the interrogation of barley germplasm there is evidence that HvMYB4H and HvMYC4H have undergone human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.

Localised expression of OsIAA29 suggests a key role for auxin in regulating development of the dorsal aleurone of early rice grains.

MAIN CONCLUSION: Non-canonical AUX/IAA protein, OsIAA29, and ZmMPR-1 homologues, OsMRPLs, are part of an auxin-related signalling cascade operating in the dorsal aleurone during early rice grain development. Endosperm of rice and other cereals accumulates high concentrations of the predominant in planta auxin, indole-3-acetic acid (IAA) during early grain development. However, IAA signalling and function during endosperm development are poorly understood. Here, we report that OsYUC12 (an auxin biosynthesis gene) and OsIAA29 (encoding a non-canonical AUX/IAA) are both expressed exclusively in grains, reaching a maximum 5-6 days after pollination. OsYUC12 expression is localised in the aleurone, sub-aleurone and embryo, whereas OsIAA29 expression is restricted to a narrow strip in the dorsal aleurone, directly under the vascular bundle. Although rice has been reported to lack endosperm transfer cells (ETCs), this region of the aleurone is enriched with sugar transporters and is likely to play a key role in apoplastic nutrient transfer, analogous to ETCs in other cereals. OsIAA29 has orthologues only in grass species; expression of which is also specific to early grain development. OsYUC12 and OsIAA29 are temporally co-expressed with two genes (AL1 and OsPR602) previously linked to the development of dorsal aleurone or ETCs. Also up-regulated at the same time is a cluster of MYB-related genes (designated OsMRPLs) homologous to ZmMRP-1, which regulates maize ETC development. Wheat homologues of ZmMRP-1 are similarly expressed in ETCs. Although previous work has suggested that other cereals do not have orthologues of ZmMRP-1, our work suggests OsIAA29 and OsMRPLs and their homologues in other grasses are part of an auxin-regulated, conserved signalling network involved in the differentiation of cells with ETC-like function in developing cereal grains.

Subcellular dynamics studies of iron reveal how tissue-specific distribution patterns are established in developing wheat grains.

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 (57 Fe) 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 57 Fe, was observed in aleurone cells and may represent iron being delivered to phytate globoids. Correlation of the 57 Fe 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.

Interaction between endogenous H2O2 and OsVPE3 in the GA-induced PCD of rice aleurone layers.

KEY MESSAGE: Endogenous hydrogen peroxide (H2O2) is involved in regulating the gibberellic acid-induced programmed cell death (PCD) of the aleurone layers by cooperating with OsVPE3 during rice seed germination. Preliminary experiments revealed that H2O2 produced by the NOX pathway is the key factor affecting rice germination. Histochemical analysis indicated that H2O2 is located in the aleurone layer. Both the H2O2 scavenger DMTU and the NOX inhibitor DPI decreased H2O2 content and significantly slowed down vacuolation in a dose-dependent manner. Interestingly, DMTU down-regulated the OsNOX8 transcript or DMTU and DPI decreased the intracellular H2O2 level, resulting in a delay of PCD. In contrast, GA and H2O2 up-regulated the OsNOX8 transcript and intracellular H2O2 level, leading to premature PCD, and the effects of GA and H2O2 were reversed by DMTU and DPI, respectively. These results showed that the imbalance of intracellular H2O2 levels leads to the delayed or premature PCD. Further experiments indicated that GA up-regulated the OsVPE3 transcript and VPE activity, and the effect was reversed by DPI. Furthermore, Ac-YVAD-CMK significantly blocked H2O2 accumulation, and DPI + Ac-YVAD-CMK had a more significant inhibitory effect compared with DPI alone, resulting in the delayed PCD, suggesting that OsVPE3 regulates PCD by promoting H2O2 generation. Meanwhile, DPI significantly inhibited the OsVPE3 transcript and VPE activity, and in turn delayed PCD occurrence, suggesting that the H2O2 produced by the NOX pathway may regulate PCD by up-regulating the OsVPE3 transcript. Thus, the endogenous H2O2 produced by the NOX pathway mediates the GA-induced PCD of rice aleurone layers by interacting with OsVPE3.

High-resolution spatiotemporal transcriptome analyses during cellularization of rice endosperm unveil the earliest gene regulation critical for aleurone and starchy endosperm cell fate specification.

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.

Mutations in the DNA demethylase OsROS1 result in a thickened aleurone and improved nutritional value in rice grains.

The rice endosperm, consisting of an outer single-cell layer aleurone and an inner starchy endosperm, is an important staple food for humans. While starchy endosperm stores mainly starch, the aleurone is rich in an array of proteins, vitamins, and minerals. To improve the nutritional value of rice, we screened for mutants with thickened aleurones using a half-seed assay and identified thick aleurone 2-1 (ta2-1), in which the aleurone has 4.8 ± 2.2 cell layers on average. Except for starch, the contents of all measured nutritional factors, including lipids, proteins, vitamins, minerals, and dietary fibers, were increased in ta2-1 grains. Map-based cloning showed that TA2 encodes the DNA demethylase OsROS1. A point mutation in the 14th intron of OsROS1 led to alternative splicing that generated an extra transcript, mOsROS1, with a 21-nt insertion from the intron. Genetic analyses showed that the ta2-1 phenotype is inherited with an unusual gametophytic maternal effect, which is caused not by imprinted gene expression but rather by the presence of the mOsROS1 transcript. Five additional ta2 alleles with the increased aleurone cell layer and different inheritance patterns were identified by TILLING. Genome-wide bisulfite sequencing revealed general increases in CG and CHG methylations in ta2-1 endosperms, along with hypermethylation and reduced expression in two putative aleurone differentiation-related transcription factors. This study thus suggests that OsROS1-mediated DNA demethylation restricts the number of aleurone cell layers in rice and provides a way to improve the nutrition of rice.

Improving the digestibility of cereal fractions of wheat, maize, and rice by a carbohydrase complex rich in xylanases and arabinofuranosidases: an in vitro digestion study.

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.