Low Molecular Weight Glutenin Research Articles

Variations in yield and gluten proteins in durum wheat varieties under late-season foliar versus soil application of nitrogen fertilizer in a northern Mediterranean environment.

With the increasing demand for high-quality foodstuffs and concern for environmental sustainability, late-season nitrogen (N) foliar fertilization of common wheat is now an important and widespread practice. This study investigated the effects of late-season foliar versus soil N fertilization on yield and protein content of four varieties of durum wheat, Aureo, Ariosto, Biensur and Liberdur, in a three-year field trial in northern Italy. Variations in low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that N applied to the canopy did not improve protein rate compared with N application to the soil (general mean 138 mg g-1 ), but moderately increased productivity in the high-yielding varieties Liberdur and Biensur (three-year means 7.23 vs 7.13 and 7.53 vs 7.09 t ha-1 respectively). Technological quality was mainly related to variety choice, Aureo and Ariosto having higher protein rates and glutenin/gliadin ratios. Also found was a strong 'variety × N application method' interaction in the proportions of protein subunits within each class, particularly LMW-GS and gliadins. A promising result was the higher N uptake efficiency, although as apparent balance, combined with higher HMW/LMW-GS ratio in var. Biensur. Late-season foliar N fertilization allows N fertilizer saving, potentially providing environmental benefits in the rainy climate of the northern Mediterranean area, and also leads to variety-dependent up-regulation of essential LMW-GS and gliadins. Variety choice is a key factor in obtaining high technological quality, although it is currently associated with modest grain yield. This study provides evidence of high quality in the specific high-yielding variety Biensur, suggesting its potential as a mono-varietal semolina for pasta production. © 2017 Society of Chemical Industry.

ITRAQ-based quantitative proteome characterization of wheat grains during filling stages

AbstractUsing isobaric tags for relative and absolute quantification (iTRAQ) and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins (DEPs) includind high-molecular weight glutenin subunit (W5AIU1), low-molecular weight glutenin subunit (Q8W3V4), gliadin/avenin-like seed protein (D2KFG9), and avenin-like protein (W5DVL2), all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Development of a set of PCR markers specific to Aegilops longissima chromosome arms and application in breeding a translocation line.

Transcriptome data were used to develop 134 Aegilops longissima specific PCR markers and their comparative maps were constructed by contrasting with the homologous genes in the wheat B genome. Three wheat- Ae. longissima 1BL·1S l S translocation lines were identified using the correspondence markers. Aegilops longissima is an important wild species of common wheat that harbors many genes that can be used to improve various traits of common wheat (Triticum aestivum L.). To efficiently transfer the traits conferred by these Ae. longissima genes into wheat, we sequenced the whole expression transcript of Ae. longissima. Using the transcriptome data, we developed 134 specific polymerase chain reaction markers located on the 14 chromosome arms of Ae. longissima. These novel molecular markers were assigned to specific chromosome locations based on a comparison with the homologous genes in the B genome of wheat. Annotation of these genes showed that most had functions related to metabolic processes, hydrolase activity, or catalytic activity. Additionally, we used these markers to identify three wheat-Ae. longissima 1BL·1SlS translocation lines in somatic variation populations resulting from a cross between wheat cultivar Westonia and a wheat-Ae. longissima substitution line 1Sl(1B). The translocation lines had several low molecular weight glutenin subunits encoding genes beneficial to flour processing quality that came from Ae. longissima 1SlS. The three translocation lines were also confirmed by genomic in situ hybridization. These translocation lines will be further evaluated for potential quality improvement of bread-making properties of wheat.

Allelic variation at high-molecular weight and low-molecular weight glutenin subunit genes in Moroccan bread wheat and durum wheat cultivars

Glutenin is a major protein fraction contributing to the functional properties of gluten and dough. The glutenin constitutes 30–40% of the protein in wheat flour and about half of that in gluten. It is essential to identify correct glutenin alleles and to improve wheat quality by selecting alleles that exert favorable effects. Moroccan wheat cultivars are unique in West Asia and North Africa region, since many of them possess resistance to Hessian fly, a pest, which is becoming important in other countries in the region. Hence, these cultivars are being used as donor for the resistance in the breeding program. Here, we determine the allelic variation in high-molecular weight glutenin subunits (HMW-GS) and low-molecular weight glutenin subunits (LMW-GS) in Moroccan cultivars of bread and durum wheat using the gene-specific PCR markers. In 20 cultivars of bread wheat, 9 different allele variants were detected at HMW-GS and 13 different allele variants were detected at LMW-GS, in which the alleles Glu-A1b (2*), Glu-B1i (17 + 18), Glu-B1c (7*/7 + 9), Glu-D1d (5 + 10), Glu-A3c, Glu-B3 h, and Glu-D3b were the most frequents. In 26 cultivars of durum wheat, less allelic variation was found: seven different allele variants at HMW-GS and six different allele variants at LMW-GS were identified, in which the major alleles were Glu-A1c (null), Glu-B1b (7 + 8), Glu-B1e (20), Glu-A3c, and Glu-B3d. The mean value of the genetic diversity for the glutenin loci was 0.502 in bread wheat and 0.449 in durum wheat. Most of the glutenin alleles carried by Moroccan bread wheat cultivars impart good bread-making quality. Most of the durum wheat glutenin alleles were related to low strength dough or poor quality and need to be improved. To improve quality of Moroccan durum wheat, essentially, Glu-A1c and Glu-B3d alleles of the genes should be replaced with the better alleles through breeding.

Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods

A multiplex ligation-dependent probe amplification (MLPA) approach is described for the simultaneous detection of DNA from four common cereal ingredients in foods: wheat, barley, rye and oats. The method uses species-specific MLPA half-probes targeting DNA fragments from the ribosomal internal space transcriber (ITS) region for the detection of barley and oats, and from the genes encoding the low molecular weight (LMW) glutenin and the ω-secalin for wheat and rye, respectively. After hybridization, the probes are ligated and amplified by polymerase chain reaction (PCR) into specific and size-differential amplicons that are simultaneously detected by capillary electrophoresis. The cereal MLPA technique showed an optimal specificity against a representative number of plant and animal species. A sensitive detection of the targets (LOD of 50 mg kg−1) was achieved in a reference model cake experimentally spiked with different levels of each wheat, barley, rye and oats seeds. MLPA applicability was assessed through the analysis of 40 commercial food products with different labeling declarations regarding the targets (contain, may contain and do not declare/gluten free), indicating the presence of non-declared cereal ingredients in some of the tested samples. MLPA results were further confirmed by simplex Taqman real-time PCR assays. The described MLPA technique is a reliable and sensitive tool for screening the presence of low amounts of wheat, barley, rye and oats in processed foodstuffs, contributing to compliance with authenticity legal requirements and protecting consumers from fraudulent commercial practices or adventitious contamination which may lead to allergic reactions associated to cereal proteins ingestion.

New insight into the function of wheat glutenin proteins as investigated with two series of genetic mutants

Among the three major food crops (rice, wheat and maize), wheat is unique in accumulating gluten proteins in its grains. Of these proteins, the high and low molecular weight glutenin subunits (HMW-GSs and LMW-GSs) form glutenin macropolymers that are vital for the diverse end-uses of wheat grains. In this work, we developed a new series of deletion mutants lacking one or two of the three Glu-1 loci (Glu-A1, -B1 and -D1) specifying HMW-GSs. Comparative analysis of single and double deletion mutants reinforced the suggestion that Glu-D1 (encoding the HMW-GSs 1Dx2 and 1Dy12) has the largest effects on the parameters related to gluten and dough functionalities and breadmaking quality. Consistent with this suggestion, the deletion mutants lacking Glu-D1 or its combination with Glu-A1 or Glu-B1 generally exhibited strong decreases in functional glutenin macropolymers (FGMPs) and in the incorporation of HMW-GSs and LMW-GSs into FGMPs. Further examination of two knockout mutants missing 1Dx2 or 1Dy12 showed that 1Dx2 was clearly more effective than 1Dy12 in promoting FGMPs by enabling the incorporation of more HMW-GSs and LMW-GSs into FGMPs. The new insight obtained and the mutants developed by us may aid further research on the control of wheat end-use quality by glutenin proteins.

Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy.

The impact of breeding on grain yields of wheat varieties released during the 20th century has been extensively studied, whereas less information is available on the changes in gluten quality associated with effects on the amount and composition of glutenins and gliadins. In order to explore the effects of breeding during the 20th century on gluten quality of durum wheat for processing and health we have compared a set of old and modern Italian genotypes grown under Mediterranean conditions. The better technological performance observed for the modern varieties was found to be due not only to the introgression of superior alleles of high (HMW-GS) and low molecular weight (LMW-GS) glutenin subunits encoded at Glu-B1 and Glu-B3 loci, but also to differential expression of specific storage proteins. In particular, the higher gluten index observed in modern genotypes was correlated with an increased glutenin/gliadin ratio and the expression of B-type LMW-GS which was, on average, two times higher in the modern than in the old group of durum wheat genotypes. By contrast, no significant differences were found between old and modern durum wheat genotypes in relation to the expression of α-type and γ-type gliadins which are major fractions that trigger coeliac disease (CD) in susceptible individuals. Furthermore, a drastic decrease was observed in the expression of ω-type gliadins in the modern genotypes, mainly ω-5 gliadin (also known as Tri a 19) which is a major allergen in wheat dependent exercise induced anaphylaxis (WDEIA). Immunological and 2DE SDS-PAGE analyses indicated that these differences could be related either to a general down-regulation or to differences in numbers of isoforms. Lower rainfall during grain filling period was related to overall higher expression of HMW-GS and ω-gliadins. In conclusion, breeding activity carried out in Italy during the 20th century appears to have improved durum wheat gluten quality, both in relation to technological performance and allergenic potential.

Optimising durum wheat cultivation in North Italy: understanding the effects of site-specific fertilization on yield and protein content

Variable rate fertilization and precision harvesting could increase the potential for meeting durum wheat quality standards. Field spatial distribution of yield and protein content, and their interactions with soil properties and N fertilization were evaluated in an experiment on durum wheat in North Italy in 2011 and 2012. Variable rate fertilization was adopted in three management zones (MZs) with increasing soil fertility, and a foliar N was applied at flowering to investigate differences in protein quantity and quality. During the crop cycle, changes in crop biomass and N status were monitored through NDVI measurement, and grain was sampled in each MZ and gluten proteins extracted at harvest. Spatial variability of yield and protein content was mainly driven by soil texture and base fertilization in both the years, while foliar fertilization was not efficient in enhancing grain protein content. Variable rate fertilization partially mitigated the weather impact; however, unpredictable weather conditions resulted in low N use efficiency. High N rates were confirmed to provide high protein levels and enhance gluten proteins technological quality, but with a risk for the environment. The marked spatial variability in grain quality in terms of total protein and gluten protein content, and the ratio between glutenin/gliadin and high and low-molecular weight glutenin sub-units, suggested the implementation of zone harvesting as a strategy to exploit the positive interaction between grain quality and soil fertility.

Evidence of intralocus recombination at the Glu-3 loci in bread wheat (Triticum aestivum L.).

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci. The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

Definition of the low molecular weight glutenin subunit gene family members in a set of standard bread wheat (Triticum aestivum L.) varieties

Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the viscoelastic properties of wheat dough. The LMW-GSs are encoded by multi-gene families at the Glu-A3, Glu-B3 and Glu-D3 loci, with more than 15 genes present in most bread wheat varieties. However, the genic profile associated with different alleles has not been clearly defined. Here, the LMW-GSs in a set of standard varieties were analyzed using molecular markers. In most cases, each Glu-3 allele was represented by a specific haplotype; however, some alleles were undistinguishable. The Glu-A3e and Glu-A3g alleles showed an identical marker haplotype, as did the alleles Glu-B3c and Glu-B3d, and Glu-B3f and Glu-B3ab. In contrast, two haplotypes among varieties designated Glu-D3c were differentiated. The marker profiles present at the Glu-D3 locus exhibited less variation compared to the genes at the Glu-A3 and Glu-B3 loci. Results show the potential of the LMW-GS gene marker system in the characterization of the LMW-GS alleles present in specific bread wheat varieties, and its reconciliation with protein-based nomenclature. This approach will advance the understanding of the contribution of each of the LMW-GS gene alleles in the control of the end-use quality.

Identification and molecular characterization of novel LMW-m and -s glutenin genes, and a chimeric -m/-i glutenin gene in 1A chromosome of three diploid Triticum species

Low molecular weight glutenin subunits (LMWGs) are part of the gluten network that gives dough its viscoelastic properties. A-genome-containing diploid species related to modern polyploid wheat show great variability in these subunits. The current study characterized the variability of LMW-m and -s genes, being identified fifteen LMWGs genes in the three species evaluated, of which 14 were novel. Ten were pseudogenes, which are common in cereal prolamins. The rest of genes, with intact coding region, corresponded to the LMW-m genes and some single nucleotide polymorphisms and insertion/deletion events were detected, which could alter protein structure and affect dough quality. Two variants of the LMW-s genes were detected in cultivated einkorn and Triticum urartu, the former being novel. The LMW-m and -s genes were related to TuA3-391 and TuA3-400 genes, and to the Tu-460 gene, respectively, of T. urartu. Screening the sequences characterized for reactive epitopes of celiac disease revealed that LMW-m could be less toxic than other subunits for celiac patients. One novel chimeric gene with features from LMW-m and LMW-i genes was detected in T. urartu, and it produces a novel mature protein that may have a distinguishing effect on dough quality. Novel insights into the evolution of LMWGs genes are also reported. These species are a potential source of novel LMWGs variants.

Effect of glutenin subunits on the baking quality of Brazilian wheat genotypes

This study aimed to evaluate the effect of the high and low molecular weight glutenin subunits on the grain traits of sixteen Brazilian wheat genotypes. Grain hardness index, milling traits, physicochemical and rheological properties of the flour, and specific volume and firmness of the bread were evaluated. Physicochemical properties of the flour were not influenced by glutenin subunits. Genotypes with subunits at the Glu-B1 (17+18 or 7+8), Glu-D1 (5+10), and Glu-A3 (b) were associated with strong flours and bread with high specific volume and low firmness. The subunits at the Glu-A1 and Glu-B3 had no effect on the rheological properties of the dough and bread quality, while the subunit 2+12 at Glu-D1 negatively affected the resistance to extension, and specific volume and firmness of the bread. Specific volume and firmness of the bread were influenced by the rheological properties of the dough, while the flour protein content was not important to define wheat quality. The identification of glutenin subunits at different loci along with the rheological tests of the flour are fundamental in estimating the potential use of different materials developed in wheat breeding.

Impact of Elevated CO<sub>2</sub> on Wheat Growth and Yield under Free Air CO<sub>2</sub> Enrichment

Impact of elevated CO2 (free air CO2 enrichment) was studied on wheat (Triticum aestivum L. var Kundan) growth, yield and proteome. Elevated CO2 significantly impacted both underground (+24%) and aboveground (+15%) biomass. Grain weight/plant and harvest index were increased by 35% and 11.4%, respectively under high CO2. On the other hand, seed protein content was decreased by 19% under CO2 enrichment while seed starch and soluble sugar contents were increased by 8% and 23%, respectively. Wheat leaf proteomics revealed that 50 proteins were showing differential expression. Twenty proteins were more abundant while 30 were less abundant. Thirty two proteins were identified by MALDI TOF TOF. More abundant proteins were related to defense, photosynthesis, energy metabolism etc. While less abundant proteins were related to glycolysis and gluconeogenesis. Wheat grain proteomics revealed that out of 49 differentially abundant proteins, 24 were more in abundance and 25 were less in abundance in wheat grains under eCO2 condition. Thirty three proteins were identified and functionally characterized. They were found to be involved mainly in carbon metabolism, storage, defence and proteolysis. Gluten proteins are the major component of wheat storage proteins. Our results showed that both high and low molecular weight glutenins were more in eCO2 wheat seeds while there was no change in gliadin evels. This might alter wheat dough strength. Concentration of grain Cr and As was increased at eCO2 while that of Fe, Cu, Zn and Se were found to be decreased. Dynamics of carbon utilization and metabolic abilities of soil microbes under eCO2 were significantly altered. Our study showed that altered wheat seed composition is cause for concern vis-a-vis nutrition and health and for industries which may have implications for agriculturally dominated country like India.

Allelic analysis of low molecular weight glutenin subunits using 2-DGE in Korean wheat cultivars.

Two-dimensional gel electrophoresis (2-DGE) was used as a complement to SDS-PAGE to determine the allelic compositions of LMW-GS in 32 Korean wheat cultivars. Protein patterns generated by 2-DGE from each cultivar were compared to patterns from standard wheat cultivars for each allele. At the Glu-A3 locus, thirteen c, twelve d, three e (null), two g and two new alleles were identified. At the Glu-B3 locus, one b, nineteen d, four h, one i and five ad alleles were identified. At the Glu-D3 locus, twenty-three a, four b, four c and one l alleles were identified. When compared to results obtained previously using SDS-PAGE, there were discrepancies in the allelic designations of 10 of 32 cultivars (31%). While SDS-PAGE is a rapid and relatively simple method for assessing LMW-GS composition, the similar mobilities of the proteins makes it difficult to discriminate certain alleles. 2-DGE is a more complicated technique, but provides a more accurate picture of the complement of the LMW-GS in a given cultivar. In addition to providing essential information for wheat breeders, the 2-DGE reference maps generated in this study will make it possible to study the contributions of individual LMW-GS to flour quality.

Effects of glu-1 and glu-3 allelic variations on wheat glutenin macropolymer (GMP) content as revealed by size-exclusion high performance liquid chromatography (SE-HPLC)

Gluten macropolymers (GMPs), formed by seed storage proteins glutenins and gliadins through intermolecular disulfide bond, confer dough viscoelasticity andwheat processing quality. Glutenins consist of highand low molecular weight glutenin subunits (HMW-GS, LMW-GS)encoded by Glu-1 and Glu-3 loci, respectively. The allelic variations at both loci have important effects on GMP content and breadmaking quality. In this study, GMP extraction and size-exclusion high performance liquid chromatography (SE-HPLC) separation were optimized, and then applied to investigate the effects of Glu-1 and Glu-3 allelic variations on GMP content using different wheat genotypes, chromosome substitution lines and near-isogenic lines (NILs). The results showed that the optimized GMP extraction and SE-HPLC protocol could obtain a reproducible separation and reliable quantitation of GMP content with small samples. The allelic variations at Glu-1 and Glu-3 were closely related to GMP content. Particularly, Glu-D1 dencoded 1Dx5+1Dy10 subunits, Glu-A3a and Glu-B3 hencoded an abundant LMW-B subunit respectively had positive effects on GMP content and bread making quality. The introgress of HMW-GS and LMW-GS into bread wheat from related genomes could significantly increase GMP content, indicating that wheat related species has potential gene resources for breadmaking quality improvement. Our results demonstrated that SE-HPLC could serve as an effective tool for rapid separation and quantitation of GMPs and had potential application for gluten quality screening in the early generations during wheat quality improvement program.

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.

Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome

DH20, a new wheat mutant missing low-molecular weight glutenin subunits encoded by the Glu-B3 locus, was discovered among double haploid lines obtained from a cross between the Korean wheat cultivars Keumkang and Olgeuru. Absence of the Glu-B3 LMW-GS proteins was determined by one-dimensional gel electrophoresis (SDS-PAGE) and confirmed by two-dimensional gel electrophoresis (2-DGE) and tandem mass spectrometry (MS/MS). The deletion of Glu-B3 genes was also demonstrated using allele specific DNA markers. Basic agronomic traits, protein content, dough mixing properties and bread loaf volume of DH20 and parental wheat cultivars were evaluated in field-grown wheat over a two year period. This mutant is a valuable resource for understanding the function of individual LMW-GS alleles or genes at the Glu-B3 locus using either breeding or biotechnology approaches.

Identification and molecular characterization of one novel 1SPl-encoded s-type low molecular weight glutenin B-subunit from 1Sl(1B) substitution line of wheat variety Chinese Spring (Triticum aestivum)

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Determination of wheat bug (Eurygaster spp.) damage in durum wheat (Triticum durum L.) by electrophoresis and rapid visco analyser

The degradation effects of wheat bug protease(s) on glutenin proteins of durum wheat cultivars were investigated by electrophoresis and modified rapid visco analyser (RVA) test. Glutenin patterns of the bug damaged durum wheats changed substantially due to bug protease(s). Although high molecular weight glutenin subunits (HMW-GS) of three cultivars (Ege, Svevo, and Zenith) disappeared after 60 min of incubation, the HMW-GS of other two cultivars (Diyarbakir and Firat) were still visible even after the longest incubation period at medium damage level. It shows that there was an intercultivar variation in susceptibility to hydrolysis by bug proteolytic enzymes. Low molecular weight glutenin subunits of all cultivars decreased substantially after 30 min of incubation. The RVA curves indicated a clear reduction in viscosity in semolina samples with both medium and high damage levels as compared to their respective undamaged (control) samples. There were significant correlations (p < 0.001) between bug damage level and viscosities at 3 min (r = −0.765), at 4.5 min (r = −0.549) and at 10 min (r = −0.835), breakdown value (r = −0.534) and decay rate (r = 0.600). Consequently, hydrolysis rate of wheat bug protease(s) can be determined by modified RVA technique without much more chemicals, procedures and expensive equipments.

Synthesis and accumulation of glutenin subunits during grain development in bread wheat as revealed by reversed-phase ultra-performance liquid chromatography (RP-UPLC)

Wheat glutenins containing high and low molecular weight glutenin subunits (HMW-GS and LMW-GS) are the major determinants of wheat gluten quality. In this study, the recently developed reversed-phase ultra-performance liquid chromatography (RP-UPLC) was used to study the synthesis and accumulation patterns of glutenins during grain development of four Chinese bread wheat cultivars with different gluten quality. Developing grains were collected based on thermal times from 150 °Cd to 750 °Cd at 100 °Cd intervals, and the content of glutenin subunits and their accumulation patterns were determined by RP-UPLC as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that HMW-GS and LMW-GS synthesis were initiated currently at 250 °Cd and they displayed a gradually upregulated expression. All the HMW-GS can be detected at 250 °Cd, earlier than LMW-GS. Different glutenin subunits and genotypes showed clear accumulation diversity during grain development. Particularly, 1Dx5 + 1Dy10 in the cultivar Gaocheng 8901 and Zhongyou 9507 with superior dough properties were accumulated faster at early stages than 1Dx2 + 1Dy12 in Jingdong 8 and Zhengmai 9023 with poor dough quality, suggesting that faster accumulation rate of glutenin proteins at the early stages of grain development may contribute to the formation of superior gluten structure and dough quality.