Low Molecular Weight Glutenin Subunits Research Articles

Proteomic Analysis of Proteins Responsive to Drought and Low Temperature Stress in a Hard Red Spring Wheat Cultivar.

Drought stress is becoming more prevalent with global warming, and has been shown to have large effects on gluten proteins linked to wheat bread making quality. Likewise, low temperature stress can detrimentally affect proteins in wheat. This study was done to determine the differential abundance of high molecular weight (HMW) glutenin proteins in a drought and low temperature stressed high quality hard red spring wheat cultivar (PAN3478), against a control. The treatments were applied in the greenhouse at the soft dough stage. HMW glutenin proteins were extracted from the flour, and were separated by using two-dimensional gel electrophoresis. Protein spots that had p values lower than 0.05 and fold values equal to or greater than 1.2 were considered to be significantly differentially abundant. These proteins were further analyzed by using tandem mass spectrometry. There was a 1.3 to 1.8 fold change in 17 protein spots due to the cold treatment. The drought treatment caused a 1.3 to 3.8 fold change in 19 protein spots. These spots matched either HMW or low molecular weight (LMW) glutenin subunits. In the latter case, the C subunits of LMW glutenins were notably found to be up-regulated under both stress conditions. All the proteins that have been identified can directly influence dough characteristics. Data are available via ProteomeXchange with the identifier PXD017578.

Allelic Variation for Prolamins in Spanish Durum Wheat Landraces and Its Relationship with Quality Traits

Wheat landraces are currently being explored mainly as a source of allelic variation related to crop resilience and low-input adaptation. Characterization of their high- and low-molecular weight glutenin subunits can aid breeders to select as donor local materials those ensuring good end-use properties in the derived elite cultivars. By using protein electrophoretic methods, we have determined the prolamin allelic profile of 116 Spanish durum wheat landraces. Their quality properties (as defined by grain protein content, sodium dodecyl sulfate (SDS) sedimentation volume and mixograph behavior) have also been assessed. The study has identified six novel glutenin alleles plus some other rare alleles some of which have been associated with improved durum wheat quality. Most of the novel variation detected needs to be characterized in a wider sample of varieties to establish any eventual beneficial effect on functional quality. Further analysis of the quality properties associated to specific allele combinations of Glu-A3, Glu-B3, and Glu-A1 has disclosed some clues on the influence on quality of certain non-allelic interactions between these main prolamin-encoding loci. Some of the landraces, showing outstanding values for the gluten quality parameters analyzed, might be directly used by farmers interested in the cultivation of traditional varieties for specialized food markets.

Effect of wheat grain protein composition on end-use quality.

The quality of wheat products has been a new challenge next to wheat production which was achieved substantially during green revolution. The end-use quality of wheat is an essential factor for its commercial demand. The quality of wheat is largely based on the wheat storage proteins which extensively influences the dough properties. High molecular weight glutenin subunits (HMWGS), low molecular weight glutenin subunits (LMWGS) and gliadins significantly influence the end-use quality. Genomics and proteomics study of these gluten proteins of bread and durum wheat have explored new avenues for precise identification of the alleles and their role in end-use quality improvement. Secalin protein of Secale cereale encoded by Sec-1 loci and isassociated with 1RS.1BL translocation has been known for deterioration of end-use quality. Chromosomal manipulations using various approaches have led to the development of new recombinant lines of wheat without secalin. Advanced techniques associated with assessment of end-use quality have integrated the knowledge of useful or deteriorating HMWGS/LMWGS alleles and their potential role in end-use quality. This review gives a comprehensive insight of different aspects of the end-use quality perspective for bread making in wheat along with some information on the immunological interference of gluten in celiac disease.

Change in physicochemical characteristics and molecular weight distribution of glutenin macropolymer induced by postharvest wheat maturation

The present study was aimed to investigate change in physicochemical characteristics and glutenin macropolymer (GMP) molecular weight distribution during postharvest wheat maturation. The freshly harvested wheat was stored under four different conditions (WT1, WT2, WT3 and WT4) for specified times. During maturation, the strengthening of wheat gluten structure was observed by increase in gluten index, swelling index of glutenin and sodium dodecyl sulphate (SDS) sedimentation value. The postharvest maturation resulted in contents increase of glutenin, glutenin/gliadin ratio, and GMP, and the decline of gliadin content. The contents of free sulfhydryl in GMP reduced, and S-S content rose during the whole maturation. Visible aggregates of GMP were observed in SDS-PAGE patterns for WT3 and WT4 samples after 4 weeks of storage. Moreover, in these two samples, polymerisation of gluten proteins into GMP was observed in size-exclusion high performance liquid chromatography patterns during storage. Some low molecular weight glutenin subunits (LMW-GS) incorporated into the aggregate, or combination of high molecular weight glutenin subunits and LMW-GS formed large polymeric glutenins by S-S linkage, and then assembled into GMP in postharvest wheat maturation. GMP polymerisation probably results in the improvement of wheat quality.

The Genetic Potential of a Germplasm of Interspecific Crosses between Durum Wheats (Triticum turgidumL. ssp.durum(Desf.) Husn.) and their Relatives (T. dicoccumSchübl. andT. polonicumL.) in Five Glutenin Loci

Wheat endosperm storage proteins are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 5 glutenin loci were identified from a set of 38 tetraploid wheat germplasm obtained from interspecific crosses between durum wheats (Triticum turgidum L. ssp. durum (Desf.) Husn.) and their relatives (T. dicoccum Schubl. and T. polonicum L.) using SDS-PAGE. At Glu-A1 and Glu-B1, encoding high molecular weight glutenin subunits (HMW-GS), 2 and 4 alleles were observed, respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 3, 5 and 3 alleles were identified at loci Glu-A3, Glu-B3 and Glu-B2, respectively. One new allele was detected at Glu-B3 locus and appeared in nine accessions obtained from five crosses. This allele codes for five subunits (2 + 8 + 9 + 13 + 18), encoded by the Glu-B3b without subunit 16 plus subunits 2 and 18. A total of 38 patterns resulted from the genetic combination of the alleles encoding at the five glutenin loci. This led to a significantly higher Nei coefficient of genetic variation in Glu-1, Glu-3 and Glu-B2 loci (0.54). The germplasm analyzed exhibited allelic variation in HMW and LMW glutenin subunit composition and the variation differed from that of tetraploid wheats of other countries. The presence of high quality alleles in glutenin loci have led the accessions to be considered as an asset in breeding programs aimed for wheat quality.

Enriching LMW-GS alleles and strengthening gluten properties of common wheat through wide hybridization with wild emmer.

Two advanced lines (BAd7-209 and BAd7-213) with identical high-molecular-weight glutenin subunit composition were obtained via wide hybridization between low-gluten cultivar chuannong16 (CN16) and wild emmer D97 (D97). BAd7-209 was better than BAd7-213, and both of them were much better than CN16 in a dough quality test. We found that BAd7-209 had more abundant and higher expression levels of low-molecular-weight glutenin subunit (LMW-GS) proteins than those of BAd7-213. Twenty-nine novel LMW-GS genes at Glu-A3 locus were isolated from BAd7-209, BAd7-213 and their parents. We found that all 29 LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Twenty-seven genes encode LMW-m-type subunits, and two encode LMW-i-type subunits. BAd7-209 had a higher number of LMW-GS genes than BAd7-213, CN16, and D97. Two wild emmer genes MG574329 and MG574330 were present in the two advanced lines. Most of the LMW-m-type genes showed minor nucleotide variations between wide hybrids and their parents that could be induced through the wide hybridization process. Our results demonstrated that the wild emmer LMW-GS alleles could be feasibly transferred and integrated into common wheat background via wide hybridization and the potential value of the wild emmer LMW-GS alleles in breeding programs designed to improve wheat flour quality.

Effect of water-extractable arabinoxylan with different molecular weight on the heat-induced aggregation behavior of gluten

Water-extractable arabinoxylan (WEAX) can improve the cereal food quality by enhancing gluten characteristics. Although the effect of WEAX on gluten quality at the dough formation stage has been extensively studied, interactions between WEAX and gluten upon the cooking stage remain far more to be revealed. In this work, the effect of WEAX with same branching degree but varied in molecular weight (Mw) on the heat-induced aggregation behavior of gluten was comparatively studied. The results showed that addition of WEAX, especially low Mw WEAX (L-WEAX) could improve the viscoelasticity of gluten during heating. WEAX induced higher enlarged degree of particle size and aggregation trend of gluten upon heating, indicating that WEAX promoted the thermal aggregation of gluten. Moreover, gluten with L-WEAX was more likely to aggregate than that with high Mw WEAX (H-WEAX). The enhanced aggregation ability of HMW-glutenin subunit x-type, LMW glutenin subunits (mainly i- and s-type), γ-gliadin and CSB_α gliadin 9/18 contributed to the promoted heat-induced aggregation behavior of gluten by WEAX. This study can provide a theoretical basis for the exploitation of WEAX as a high-efficiency dough improver.

Changes in Protein Composition in the Grain and Malt after Fusarium Infection Dependently of Wheat Resistance.

The grain yield, as well as the quality and safety of the wheat grains and corresponding malt can be compromised by Fusarium spp. infection. The protein content of the grain affects the chemical composition and enzyme levels of the finished malt. The malting industry demands varieties with good malting and brewing performance, as well as good agronomic performance and disease resistance. The best method of disease control is breeding and selection for resistant varieties. Due to higher requirements for malting wheat worldwide, the goal of this investigation was to explore changes in protein distribution in wheat grains and corresponding malt, which are under higher pressure of Fusarium head blight (FHB) infestation in field conditions. The present study provides new knowledge on the impact of the FHB on the distribution of protein components of naturally Fusarium-infected (control) and Fusarium-inoculated wheat varieties in the grain and the corresponding malt in two consecutive years (2015/2016 and 2016/2017). The results showed that Fusarium infection of the susceptible variety Golubica, decreased total glutenins (5.9%), and both high and low molecular weight glutenin subunits (2.5% and 3.5%, respectively) in wheat grains, compared to control, in 2016. In contrast, gliadins and α-gliadins increased significantly (+7.6% and +5.1%, respectively) in the same variety. Wheat grains of the more resistant variety Vulkan showed an increase of the total glutenins content (+4.3%), and of high and of low molecular weight glutenin subunits (+1.2% and +3.2%, respectively) after Fusarium-inoculation, compared to naturally infected grains in 2016. Susceptible variety Golubica increased total glutenins (+9.1%), and both high and low molecular weight glutenin subunits (+3.5% and +5.6%, respectively) after Fusarium-inoculation in wheat malt, compared to naturally infected malt in 2016. In 2017, when disease pressure was higher than in 2016, there was a tendency in all varieties to increase gliadins and its sub fractions after malting, and to decrease glutenins and its sub fractions in Fusarium-inoculated treatment. In conclusion, FHB dramatically depressed grain yield (up to 37%) and quality (glutenins and high molecular weight subunits) in the susceptible Fusarium variety, which makes it inconvenient for malting.

Genotype x environment interaction and genetic gain for grain yield and grain quality traits in Turkish spring wheat released between 1964 and 2010.

The study was conducted to determine the effects of genotype (G), environment (E), their interaction (GEI) and genetic gain on yield and grain quality traits in Turkish spring wheat cultivars released between 1964 and 2010. We conducted a multi-environment trial at three testing locations: Adana, Adapazarı, and Izmir, during the 2009, 2011 and 2013 cropping seasons and tested 35 cultivars released by the respective breeding programs. Allelic variations of high and low molecular weight glutenin subunits (HMW-GS and LMW-GS) and 1B/1R translocation was also determined and evaluated in all cultivars. Comparing yield across three locations, Adana (6416 kg ha-1) yield was relatively higher than in Izmir (5887 kg ha-1) and Adapazarı (5205 kg ha-1) (P<0.001). Overall, GY was influenced by the varieties, testing location and breeding programs (P<0.001). Cultivars from Izmir breeding program performed relatively better (6174 kg ha-1) than those from Adana (5996 kg ha-1) and Adapazarı (5351 kg ha-1) (<0.001). We recommend Ziyabey-98, Menemen, and Basribey-95, for stable grain yield in spring wheat production across the studied regions because of their wide adaptability, and Pamukova-97 for future breeding to improve grain quality parameters. We found three breeding programs have successfully increased the grain yield and quality traits for 46 years. As a group, cultivars released after 2000 had the highest yield indicating breeding progress. Genetic gain for GY was 30.9 kg ha−1 per year from 1964 with annual increase compared to the yield of older cultivar Akova B-2 (4102 kg ha-1) which constitutes a 0.75% rate of genetic gain. Improvement in grain quality was related to change in protein composition rather than an increase in protein content whereas yield improvement seems to mainly be related to increases in test weight and 1000 kernel weight. High molecular weight glutenin subunit (HMW-GS) 5+10 showed an increase in frequency whereas 2+12 showed a decrease over the breeding period.

Rheological and microstructural characteristics of low molecular weight glutenin subunits of commercial wheats

A study was conducted on the effects occurred in rheological properties of base flour dough by the addition of gluten, glutenin and purified low molecular weight glutenin subunits (LMW-GS) using a 4 g sample Microdoughlab (MDL). Incorporation of these elements brought about a significant increase in the dough strength in the order of LMW-GS < gluten < glutenin. LMW-GS from variety C 306 brought a decrease in the dough development time (DDT; 2.03 min), dough stability (DS; 3 min) and peak energy (EP; 2.90 Wh/kg) values. On the contrary, the effects of LMW-GS extracted from variety PBW 550 were more strong as indicated by an increase in DDT (2.75 min), DS (3.30 min) and EP (4.20 Wh/kg). The alterations in the microstructure of dough by the inclusion of gluten, glutenin and LMW-GS, which lacks resemblance among different samples, were contemplated subjecting it to Scanning Electron Microscopy (SEM).

Elimination of Omega-1,2 Gliadins From Bread Wheat (Triticum aestivum) Flour: Effects on Immunogenic Potential and End-Use Quality.

The omega-1,2 gliadins are a group of wheat gluten proteins that contain immunodominant epitopes for celiac disease (CD) and also have been associated with food allergies. To reduce the levels of these proteins in the flour, bread wheat (Triticum aestivum cv. Butte 86) was genetically transformed with an RNA interference plasmid that targeted a 141 bp region at the 5′ end of an omega-1,2 gliadin gene. Flour proteins from two transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry. In one line, the omega-1,2 gliadins were missing with few other changes in the proteome. In the other line, striking changes in the proteome were observed and nearly all gliadins and low molecular weight glutenin subunits (LMW-GS) were absent. High molecular weight glutenin subunits (HMW-GS) increased in this line and those that showed the largest increases had molecular weights slightly less than those in the non-transgenic, possibly due to post-translational processing. In addition, there were increases in non-gluten proteins such as triticins, purinins, globulins, serpins, and alpha-amylase/protease inhibitors. Reactivity of flour proteins with serum IgG and IgA antibodies from a cohort of CD patients was reduced significantly in both transgenic lines. Both mixing time and tolerance were improved in the line without omega-1,2 gliadins while mixing properties were diminished in the line missing most gluten proteins. The data suggest that biotechnology approaches may be used to create wheat lines with reduced immunogenic potential in the context of gluten sensitivity without compromising end-use quality.

Unravelling the hidden inter and intra-varietal diversity of durum wheat commercial varieties used in Portugal

AbstractAssessing durum wheat genomic diversity is crucial in a changing environmental particularly in the Mediterranean region where it is largely used to produce pasta. Durum wheat varieties cultivated in Portugal and previously assessed regarding thermotolerance ability were screened for the variability of coding sequences associated with technological traits and repetitive sequences. As expected, reduced variability was observed regarding low molecular weight glutenin subunits (LMW-GS) but a specific LMW-GS allelic form associated with improved pasta-making characteristics was absent in one variety. Contrastingly, molecular markers targeting repetitive elements like microsatellites and retrotransposons – Inter Simple Sequence Repeat (ISSR) and Inter Retrotransposons Amplified Polymorphism (IRAP) – disclosed significant inter and intra-varietal diversity. This high level of polymorphism was revealed by the 20 distinct ISSR/IRAP concatenated profiles observed among the 23 individuals analysed. Interestingly, median joining networks and PCoA analysis grouped individuals of the same variety and clustered varieties accordingly with geographical origin. Globally, this work demonstrates that durum wheat breeding strategies induced selection pressure for some relevant coding sequences while maintaining high levels of genomic variability in non-coding regions enriched in repetitive sequences.

Characterization of Pakistani wheat germplasm for high and low molecular weight glutenin subunits using SDS-PAGE

Characterization of Pakistani wheat germplasm for high and low molecular weight glutenin subunits using SDS-PAGE

Effects of waterlogging during grain filling on yield components, nitrogen uptake and grain quality in bread wheat

Waterlogging stress frequently affects wheat production in the current conditions. The aim of this work was to evaluate the effect of waterlogging during grain filling on grain yield components, nitrogen uptake and partitioning and gluten composition and quality in bread wheat. Two greenhouse experiments were conducted under contrasting environmental conditions in Azul, Buenos Aires, in a completely randomized design with three replicates. The cultivar chosen was Klein Leon and the waterlogging treatment was imposed from 5 days after anthesis to maturity. The effects of waterlogging during grain filling in wheat depended on explored environmental conditions: early sowing vs. late sowing. Waterlogging had not significant effects on the most variables at early sowing conditions. However, the delaying in sowing date (higher temperature and radiation) enhance the effects of waterlogging: i) reducing grain weight by 41% and total nitrogen uptake by 51%; ii) reducing the ratio between the contents of high and low molecular weight glutenin subunits, affecting gluten composition; and iii) increasing the sodium dodecyl sulfate test from 79 to 108 mm, which correlates positively with the gluten strength. Reductions in grain weight due to waterlogging during grain filling affect the milling quality, although changes in protein composition may increase or maintain the gluten strength (SDSS) under particular conditions.

Splitting nitrogen applications improves wheat storage protein composition under low N supply

AbstractFor the baking quality of wheat flours, the composition and concentration of grain protein are crucial. It is common practice to use late nitrogen (N) application to increase grain protein concentration (GPC) and hence, improve baking quality. However, the use of N fertilizer—particularly shortly before harvest—involves environmental risks. With the suitability of GPC as a parameter for baking quality predictions being more and more questioned, there are further investigations needed considering not only the GPC but also the composition of grain protein. Gluten protein composition varies depending on genotype and environmental factors, such as weather conditions and fertilization rate. To examine whether the effect of a split N application varies under different amounts of total N supply, this study investigates the effects of split nitrogen application on grain protein concentration and composition of wheat (Triticum aestivum L. cv. JB Asano) at four different N fertilization levels (0.8; 1.0; 1.2, and 1.4 g N pot−1) in a pot experiment. The GPC was affected by both, N fertilization level and split N application. In this experiment, the minimum GPC of 13%, which is required for class A wheat varieties, was only achieved when N supply was moderate (at least 1.2 g N per pot). Considering the storage protein composition, the split N application influenced the proportion of α‐/β‐gliadins and γ‐gliadins, the alterations being inconsistent. The ratio of high molecular weight (HMW) to low molecular weight (LMW) glutenin subunits was increased by the split N application only at the lowest N fertilization level. It is concluded that splitting N fertilization into three doses and hence applying one dose of N late in the season can still be a useful approach to improve GPC as well as protein composition – especially when the total N supply is low.

Effect of Glu‐D1 gene introgression and amylose content on breadmaking potential of blends of durum and hexaploid wheat

AbstractBackground and objectivesDurum wheat is used to make leavened bread; however, durum bread has inferior loaf volume, structure, and texture compared to bread made from common wheat. One approach to overcome this is to transfer key storage protein genes present at the Glu‐D1 locus from bread wheat into durum. Durum wheat Svevo missing Glu‐B1subunits 7 + 8 and Lira biotypes with low molecular weight glutenin subunits types 1 and 2 were evaluated for their breadmaking potential with and without high molecular weight glutenin subunits 2 + 12 or 5 + 10.FindingsBread made from blends of durum and a commercial baker's flour (10%, 25%, 50% w/w) assessed over two seasons in 10 different genotypes showed that as more durum was included in the mixture, loaf volume and texture score declined. Incorporation of the 2 + 12 subunit pair in the genotypes Lira42 and Lira45 improved bread quality but not in Svevo, whereas including 5 + 10 improved bread quality of Lira42 had no effect on Lira45 but reduced quality of Svevo. Low amylose Svevo had similar loaf quality to Svevo while adding 5 + 10 had minimal impact except at 50% with a small improvement in loaf quality. Bread stored up to 7 days became firmer partly due to increased starch retrogradation, and loaves were similar to bread made from baker's flour. Low amylose Svevo kept the loaf fresher but only up to 3 days of storage. Subunit pair 5 + 10 made the loaf firmer after 7 days compared to control.ConclusionsAddition of the 2 + 12 or 5 + 10 benefited the weaker type gluten as found in Lira 42 (LMW‐1, HMW 20) but not with stronger dough in Svevo even in the absence of HMW 7 + 8. It appears that while Glu‐D1 subunits are critical for good breadmaking in hexaploid wheat, they appear to have limited value in improving loaf volume and structure in durum bread.Significance and noveltySome improvements in bread quality can be obtained by introducing genes coding for Glu‐D1subunits 2 + 12 and 5 + 10 in durum wheat depending on the genotype, especially weak dough types, and the results presented comprise the first report of such effect.

Rheological evaluations and molecular marker analysis of cultivated bread wheat varieties of India.

The aim of this study was to screen Indian cultivated wheat varieties and list out the parameters/genes required to be improved for an end-product. Therefore, 30 Indian wheat varieties under cultivation by farmers were screened for 14 physico-chemical and rheological parameters, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for high molecular weight glutenin subunits (HMW-GS), DNA based molecular markers for low molecular weight glutenin subunits (LMW-GS) and puroindolines (Pin) genes. Based on grain texture, sedimentation value, farinographic, alveographic, HMW-GS and LMW-GS and biscuit making parameters, HS490 was found to be a highly suited for biscuit and soft wheat products. HI1563 and DBW14 were also found to possess characteristics such as low protein, low to medium SDS-sedimentation value and combination of 2*, 7+8 and 2+12 (HMW-GS). DBW14 also had LMW alleles desirable for biscuit quality. DBW14 needs to be improved for grain softness to make it suitable for biscuit quality while both grain softness and LMW alleles need to be improved for HI1563 to improve its biscuit spread factor and alveographic indices for extensible gluten. Rest varieties showed moderate to very strong gluten but the gluten lacked extensibility. Only four varieties K307, DBW39, NI5439 and DBW17 possessed high flour protein and moderately strong gluten. They had more balanced deformation energy (W) and configuration ratio (P/L) combination suggestive of strong and extensible gluten needed for raised bread making. Marker assisted backcross breeding is suggested as solution to produce end-use specific varieties where appropriate alleles at only a few loci need to be incorporated.

Overexpressing wheat low-molecular-weight glutenin subunits in rice (Oryza sativa L. japonica cv. Koami) seeds.

Genes encoding wheat low-molecular-weight glutenin subunits (LMW-GSs) that confer dough strength and extensibility were previously identified from Korean wheat cultivars. To improve low viscoelasticity of rice (Oryza sativa L.) dough caused by the lack of seed storage proteins comparable to wheat gluten, two genes, LMW03 and LMW28, encoding LMW-GSs are cloned from Korean wheat cultivar Jokyoung. The LMW genes are inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter. Transgenic rice plants expressing LMW03 or LMW28 in their seeds are generated using Agrobacterium-mediated transformation. The expression of recombinant wheat LMW-GS in the transgenic rice seeds was confirmed by SDS-PAGE and immunoblot analysis. Their accumulation in the endosperm and aleurone layers of rice seeds was observed through in situ immuno-hybridization.

Extraction and purification of low molecular weight glutenin subunits using size exclusion chromatography.

Crude glutenin of commercial Indian wheat varieties was fractionated into high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) by employing size-exclusion chromatography (SEC). The SEC profile of glutenins obtained with different buffers were discriminated effectively with respect to the quality of the proteins eluted in each peak. The most efficient separation of LMW-GS was achieved using 3M urea (pH 5.5) buffer under unreduced conditions. The chromatogram was segregated predominantly into three peaks with varied molecular weights as determined by SDS-PAGE. Peak I corresponded to a mixture of HMW-GS and LMW-GS (Mw 100-30kDa). Peak II enclosed LMW-GS specifically with molecular weights in the range of 45-35kDa. Lastly, a mixture of proteins associated with LMW-GS (Mw < 35kDa) were eluted in peak III. SEC proved to be a valuable tool in purifying LMW-GS in a functionally active state.

Genomics Approach to Identify the Cause of the Missing Omega-5 Gliadin Protein in O-Free Wheat

A previous work developed and identified a new omega-5 gliadin deficient wheat line named O-free by crossing Keumkang and Olgeuru, which is nutritionally quite meaningful in that omega-5 gliadin is one of the known wheat allergens. To verify the characteristics of the O-free, we performed RNA sequencing (RNAseq) analysis of the O-free and the two parent lines (Keumkang and Olgeuru). The results of the similarity analysis with the ESTs for gliadins and glutenins showed that the O-free ESTs had no similarity with the omega-5 gliadin sequences but had similarity to other gliadins and glutenins. Furthermore, mapping results between the raw RNAseq data from the O-free and the omega-5 gliadin sequence showed a clear deletion of the N-terminal sequences which are an important signature of omega-5 gliadin. We also designed specific PCR primers that could identify omega-5 gliadin in the genomic DNA. The results showed that no omega-5 gliadin fragments were detected in the O-free. According to these results, we confirmed that the deficiency of omega-5 gliadin in the O-free is not caused by post-transcriptional or post-translational regulations such as epigenetic phenomena but by a simple deletion in the chromosome. Furthermore, we showed that the low-molecular weight glutenin subunit (LMW-GS) gene in the O-free had a single nucleotide polymorphism (SNP) causing a premature stop codon, resulting in a truncated polypeptide. We expect that the O-free line may serve as an excellent source of wheat that could prevail in the hypo-allergen wheat market, which has recently gained interest world-wide.