Wheat Endosperm Research Articles

Expression of High Molecular Weight Glutenin Subunit genes correlated with gluten content in common wheat varieties from the highland of Ecuador

In this study we analyzed the performance of three wheat varieties in relation to gluten content under high-altitude growing conditions in the Andes of Ecuador. A field experiment was conducted at 3058 meters above sea level during 2009 using adapted wheat cultivar Cojitambo, cv. Carnavalero, and cv. Sibambe. Transcript accumulations of High Molecular Weight Glutenin Subunits (HMW-GS) genes were also evaluated during grain development using qRT-PCR. We recorded the expression profile of HMW-GS genes during 41 days and showed a coordinated pattern of induction with significant higher levels at 82–86 days. Transcript accumulation of 1Dx5, 1Dy10, 1Bx7, 1Ax1, and 1By9 genes were analyzed in more details during this period. The assay highlighted the specific contribution of 1Bx7, 1Dy10, and 1Dx5 during gluten formation in Ecuadorian wheat varieties. Under Andean highlands conditions, cv. Carnavalero showed the higher values of total agglomerated protein upon hydration and higher levels of expression of particular HMW-GS genes. The data suggest a correlation between wet gluten content and HMW-GS genes expression. Our study contributes to understand gluten formation in wheat endosperm under high-altitude conditions in the Andes.

Molecular Features of Wheat Endosperm Arabinoxylan Inclusion in Functional Bread.

Arabinoxylan (AX) is a major dietary fibre component found in a variety of cereals. Numerous health benefits of arabinoxylans have been reported to be associated with their solubility and molecular features. The current study reports the development of a functional bread using a combination of AX-enriched material (AEM) and optimal commercial endoxylanase. The total AX content of bread was increased to 8.2 g per 100 g available carbohydrates. The extractability of AX in breads with and without endoxylanase was determined. The results demonstrate that water-extractable AX (WE-AX) increased progressively through the bread making process. The application of endoxylanase also increased WE-AX content. The presence of 360 ppm of endoxylanase had positive effects on the bread characteristics in terms of bread volume and firmness by converting the water unextractable (WU)-AX to WE-AX. In addition, the molecular weight (Mw) distribution of the WE-AX of bread with and without endoxylanase was characterized by size-exclusion chromatography. The results show that as the portion of WE-AX increased, the amount of high Mw WE-AX (higher than 100 kDa) decreased, whereas the amount of low Mw WE-AX (lower than 100 kDa) increased from 33.2% to 44.2% through the baking process. The low Mw WE-AX further increased to 75.5% with the application of the optimal endoxylanase (360 ppm).

Probing thermal transitions and structural properties of gluten proteins using ultrasound

To probe the thermal and structural properties of gluten proteins using ultrasound. A new ultrasonic approach for characterizing the quality of wheat gluten proteins is described. Low frequency (50kHz) longitudinal ultrasonic velocity, v L, measurements were performed on gluten samples extracted from three wheat flours differing in protein content and in wheat endosperm hardness. At room temperature, v L for gluten extracted from soft flowers (Fielder) was found to be (870±92) m/s, while for gluten extracted from extra strong flours (Glenlea) it was found to be (1,940±90) m/s. In the second set of experiments, which aimed at probing thermal properties of gluten proteins, the variation in the numerical value of v L propagating in the wet gluten was found to be substantial (about 1,000m/s) when the temperature of the gluten was raised from 20 to 90°C, and also when gluten from different flour types was investigated. A continuous structural phase transition was observed, which was different for glutens extracted from different flours. Upon cooling, the velocity also varied depending on wheat type. These experiments demonstrate that ultrasonic velocity measurements can be used as a selection tool and study changes in properties of wheat proteins, particularly the thermal transitions that are critical to the quality of end products such as noodles, pasta, and bread. It was also shown that v L is sensitive to gluten class (strength or protein content), showing the potential of such measurements as an early-generation selection tool in wheat breeding programs.

Positive Assessment of Mill Stream Endosperm Purity Using Chemical Imaging

Direct measurement of wheat endosperm content, the product of flour milling, is now a reality. The cumulative endosperm mass balance calculated from a series of unit processes provides an overall assessment of separation efficiency, which varies with adjustments made to the operational settings of the individual processing steps. A summation of endosperm concentration multiplied by the flow rates of the individual unit processes is used to calculate the endosperm mass balance. Chemical imaging and endosperm yield are included for 15 product output streams from the Hal Ross flour mill (Kansas State University [KSU]). Results, including cumulative endosperm yield obtained using the new quantitative near infrared (NIR) imaging system are stated in the context of cumulative ash and ferulic acid curves that have been reported previously for two experimental wheats. Cumulative millers' curves from previous large-scale (22 bu) millings on a 23 stream KSU pilot mill were applied for the same two experimental whea...

Adhesion properties of wheat-based particles

Specific AFM analytical methods were tested to the surface evaluation of wheat powders at micrometric scale in dry conditions. The objective was to evaluate the adhesion properties of particles based on the main wheat endosperm components (starch, protein, and arabinoxylan) that are characterized by large diversity in terms of chemical composition and particle characteristics. Experiments were conducted by using the force spectroscopy mode to determine interactions generated by the surfaces of particles that were glued on AFM tips, and two reference flat and smooth surfaces, a glass and a polysine slide. Particular care has been devoted to characterize the surfaces in contact since local contact radius is a key parameter in the interpretation of the interactions. The statistical analysis of the normalized adhesion forces have shown the sensitivity of the AFM technique for hydrophobic wheat components and their correlation with the surface tension of the particles.

Mechanism of action of puroindoline derived tryptophan-rich antimicrobial peptides

A tryptophan (Trp)-rich region in the wheat endosperm protein, puroindoline A, was previously shown to possess potent antimicrobial activity against Gram-positive and Gram-negative bacteria and this was attributed to the peptide inducing membrane instability. In the present work, the antimicrobial activity of the corresponding Trp-rich region in the puroindoline B isoform was examined and its antimicrobial activity was characterized. Unexpectedly, the puroindoline B Trp-rich peptide (PuroB) was relatively inactive compared to the related puroindoline A peptide (PuroA), despite strong sequence similarity. Using the sequence of PuroA as a template, a series of PuroB variants were synthesized and the antimicrobial activity was restored. Interestingly, all of these PuroB peptides preferentially interacted with negatively charged phospholipids, but unlike PuroA, they did not disrupt the integrity of lipid bilayers. This suggests that the primary mode of action of the PuroB peptides involves an antimicrobial target other than the bacterial membrane. Further tests revealed that all of the puroindoline derived peptides bind deoxyribonucleic acid (DNA) and block macromolecular synthesis in vivo. Based on these results, it appears that the interaction between puroindoline derived peptides and membranes is only an initial step in the mode of action and that binding to intracellular targets, such as DNA and ribonucleic acid (RNA), contributes significantly to their antimicrobial mode of action.

Farinin: Characterization of a Novel Wheat Endosperm Protein Belonging to the Prolamin Superfamily

Starch granule surface-associated proteins were separated by HPLC and identified by direct protein sequencing. Among the proteins identified was one that consisted of two polypeptide chains of 11 and 19 kDa linked by disulfide bonds. Sequencing of tryptic peptides from each of the polypeptides revealed similarities between some of the peptides and avenin-like b proteins encoded by partial cDNAs in NCBI. To identify a contiguous sequence that matched all of the peptides, contigs encoding three avenin-like b proteins were constructed from ESTs of the cultivar Butte 86. All peptide sequences were found in a protein encoded by one of these contigs that had not been identified previously. Protein and DNA sequences indicated that the two polypeptide chains were derived from a parent protein that had been cleaved at the C-terminal position of an asparagine residue. The name farinin is suggested for this protein and other avenin-like b proteins. Evolutionary relationships of the protein are discussed and a simple computer molecular model was constructed. On the basis of its sequence, the new protein was likely to be allergenic but unlikely to be active in celiac disease.

Effect of Dough Mixing on Wheat Endosperm Cell Walls

Dough-derived cell wall fragments isolated by ultracentrifugation were largely derived from the starchy endosperm, with some fragments deriving from the aleurone and outer layers, as indicated by fluorescence microscopy. Dough mixing had little effect on the structure and composition of cell wall fragments compared to thin grain sections, as determined by Fourier transform infrared (FTIR) and (1)H nuclear magnetic resonance (NMR) spectroscopy. These analyses confirmed that the fragments largely comprised water-unextractable arabinoxylan and β-glucan. FTIR microspectroscopy of dough-derived cell wall fragments prepared from five bread wheat cultivars showed that two largely comprised highly substituted arabinoxylan (cv. Manital and San Pastore), one comprised a mixture of low, medium, and highly substituted arabinoxylan (cv. Hereward), and the remaining two comprised a greater proportion of low substituted arabinoxylan (cv. Claire and Yumai 34). Yumai 34 yielded a greater mass of cell wall material, and its cell walls comprised a high proportion of medium substituted arabinoxylan. Such methods will allow for the impact of bakery ingredients and processing on endosperm cells, including the addition of xylanases, to be investigated in the future to ensure any potential health benefits arising from wheat breeding are realized in the food that reaches the consumer.

On the differences in the granular architecture and starch structure between pericarp and endosperm wheat starches

AbstractIn this study, the granular organization and structure of transitory starch from wheat pericarp was analyzed and compared to the storage starch from endosperm. Pericarp starch (PS) granules were small and spherical with fuzzy blocklets on the surface. Both starches possessed A‐type crystallinity, but the relative crystallinity and the stability of crystals were higher in PS. Iodine absorption was low in PS, but iodine slightly increased the crystallinity. PS had 14% AAM compared to 33% in endosperm starch (ES). Interestingly, the AAM (fraction II) in PS was branched as judged from the λmax value of the iodine complex, whereas ES contained linear and branched amylose. The amylopectin component (fraction I) in PS and ES mainly showed differences in the internal structure. PS amylopectin had longer chains and fewer A‐chains resulting in a less branched structure compared to ES amylopectin.

Down‐regulation of glucan, water‐dikinase activity in wheat endosperm increases vegetative biomass and yield

Down‐regulation of glucan, water‐dikinase activity in wheat endosperm increases vegetative biomass and yield

Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2-transformed yeast. The over-expression of TaHMA2 increased the elongation and decreased the seed-setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over-expression also improved root-shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over-expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over-expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.

Lab-on-a-Chip method uncertanties in determination of high-molecular-weight glutenin subunits

Polymeric wheat endosperm proteins, especially the high-molecular-weight glutenin subunits (HMW-GS), are probably the most interesting protein fraction giving the essential information about bread-making quality of wheat flour. A relatively new method that shows a great potential for a fast, reliable and automatable analysis of protein purity, sizing and quantification is microfluidic or Lab-on-a-Chip (LoaC) capillary electrophoresis. This work was aimed to explore the possibilities of implementation of LoaC method to analysis of protein samples isolated from a Serbian common wheat variety, emphasizing the steps that might bring uncertainties and affect reproducibility of obtained glutenin subunits quantitation results. A good resolution of protein bands in a molecular weight range of 14.0 to 220.0 kDa was achieved. The reproducibility of HMW-GS sizing and quantitation were good, with the average coefficient of variation values of 1.2% and 12.2%. The ratio of HMW-GS to low-molecular-weight glutenin subunits (LMW-GS) was about 20%. The investigation ruled out influences of the extract solution addition and the buffer addition steps of the applied method, as well as the individual chip influence on GS quantitation results. However, there was statistically significant difference between HMW-GS quantitation results of multi-step and one-step extraction procedures applied prior to glutenin subunits extraction step.

Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue

The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage.

Comprehensive analysis of the transcription of starch synthesis genes and the transcription factor RSR1 in wheat (Triticum aestivum) endosperm

The cDNA sequences of 26 starch synthesis genes were identified in common wheat (Triticum aestivum L.), and their transcript levels were measured using quantitative real-time RT-PCR to assess the function of individual genes and the regulatory mechanism in wheat endosperm. The expression patterns of 26 genes in wheat endosperm were classified into three groups. The genes in group 1 were richly expressed in the early stage of grain development and may be involved in the construction of fundamental cell machinery, synthesis of glucan primers, and initiation of starch granules. The genes in group 2 were highly expressed during the middle and late stages of grain development, and their expression profiles were similar to the accumulation rate of endosperm starch; these genes are presumed to play a crucial role in starch production. The genes in group 3 were scantily expressed throughout the grain development period and might be associated with transitory starch synthesis. Transcripts of the negative transcription factor TaRSR1 were high at the early and late stages of grain development but low during the middle stage. The expression pattern of TaRSR1 was almost opposite to those of the group 2 starch synthesis genes, indicating that TaRSR1 might negatively regulate the expression of many endosperm starch synthesis genes during grain development.

Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.

Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm

Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fiber and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analyzed by LC-MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.

Tensile strength and fracture of cemented granular aggregates

Cemented granular aggregates include a broad class of geomaterials such as sedimentary rocks and some biomaterials such as the wheat endosperm. We present a 3D lattice element method for the simulation of such materials, modeled as a jammed assembly of particles bound together by a matrix partially filling the interstitial space. From extensive simulation data, we analyze the mechanical properties of aggregates subjected to tensile loading as a function of matrix volume fraction and particle-matrix adhesion. We observe a linear elastic behavior followed by a brutal failure along a fracture surface. The effective stiffness before failure increases almost linearly with the matrix volume fraction. We show that the tensile strength of the aggregates increases with both the increasing tensile strength at the particle-matrix interface and decreasing stress concentration as a function of matrix volume fraction. The proportion of broken bonds in the particle phase reveals a range of values of the particle-matrix adhesion and matrix volume fraction for which the cracks bypass the particles and hence no particle damage occurs. This limit is shown to depend on the relative toughness of the particle-matrix interface with respect to the particles.

Improving the yellow pigment content of bread wheat flour by selecting the three homoeologous copies of Psy1

The yellow pigment content (YPC) of endosperm affects the quality and nutritional value of wheat grain products. Major quantitative trait loci (QTL) for endosperm YPC have been repeatedly mapped on chromosomes 7A and 7B in durum and bread wheats. The genes coding for phytoene synthase (PSY1), which is involved in the biosynthesis of carotenoids, generally co-segregate with these QTL, indicating their role in determining YPC. Here, to study the genetic factors underlying endosperm YPC in bread wheat, the sequence polymorphism of the homoeologous A, B and D copies of genes coding for PSY1, Psy-A1, Psy-B1, and Psy-D1, was studied in a worldwide core collection, which was also phenotyped for flour YPC. Seven novel alleles of Psy-A1 and two novel alleles of Psy-B1 were detected, which confirms the high level of polymorphism of these genes. Two major QTL with respective candidate genes Psy-A1 and Psy-B1 were identified in the distal region of chromosomes 7A and 7B using progeny of a cross between Apache and Ornicar, high and low YPC cultivars, respectively. Association mapping confirms the role of these genes in YPC and shows that the D copy also significantly influences this trait. These results indicate that breeders need to consider all three Psy1 copies when seeking to improve the YPC of wheat endosperm.

Proteomic and genetic analysis of wheat endosperm albumins and globulins using deletion lines of cultivar Chinese Spring

Albumins and globulins from the endosperm of Triticum aestivum L. cv Chinese Spring (CS) were analysed to establish a proteome reference map for this standard wheat cultivar. Approximately, 1,145 Coomassie-stained spots were detected by two-dimensional gel electrophoresis (2DE), 410 of which were identified using mass spectrometry and data mining. Salt-soluble endosperm proteins from 67 CS deletion lines were also separated by 2DE (four gels per line). Image analysis of the 268 2DE gels as compared to the CS reference proteome allowed the detection of qualitative and quantitative variations in endosperm proteins due to chromosomal deletions. This differential analysis of spots allowed structural or regulatory genes, encoding 211 proteins, to be located on segments of the 21 wheat chromosomes. In addition, variance analysis of quantitative variations in spot volume showed that the expression of 391 proteins is controlled by one or more chromosome bins with 262 significant increases and 196 significant decreases in spot volume. The spot volume of several proteins was increased or decreased by numerous chromosomal regions and homoeologous-like regulation was revealed for some proteins. Quantitative or qualitative variation in a total of 386 proteins was influenced by genes assigned to at least one chromosomal region, while 66 % of all stained proteins were not found to be influenced by chromosome bins. Proteomics of deletion lines can, therefore, be used to simultaneously analyse the composition and genetics of a complex tissue, such as the wheat endosperm.

Down‐regulation of Glucan, Water‐Dikinase activity in wheat endosperm increases vegetative biomass and yield

A novel mechanism for increasing vegetative biomass and grain yield has been identified in wheat (Triticum aestivum). RNAi-mediated down-regulation of Glucan, Water-Dikinase (GWD), the primary enzyme required for starch phosphorylation, under the control of an endosperm-specific promoter, resulted in a decrease in starch phosphate content and an increase in grain size. Unexpectedly, consistent increases in vegetative biomass and grain yield were observed in subsequent generations. In lines where GWD expression was decreased, germination rate was slightly reduced. However, significant increases in vegetative growth from the two leaf stage were observed. In glasshouse pot trials, down-regulation of GWD led to a 29% increase in grain yield while in glasshouse tub trials simulating field row spacing and canopy development, GWD down-regulation resulted in a grain yield increase of 26%. The enhanced yield resulted from a combination of increases in seed weight, tiller number, spikelets per head and seed number per spike. In field trials, all vegetative phenotypes were reproduced with the exception of increased tiller number. The expression of the transgene and suppression of endogenous GWD RNA levels were demonstrated to be grain specific. In addition to the direct effects of GWD down-regulation, an increased level of α-amylase activity was present in the aleurone layer during grain maturation. These findings provide a potentially important novel mechanism to increase biomass and grain yield in crop improvement programmes.