Susan B. Altenbach

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography–tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the ‘Aroona’ cultivar and 12 ‘Aroona’ near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for ‘Aroona’ and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in ‘Aroona’ and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7^OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

Background: Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. Methods: Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 173 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. Results: Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Some of these also contained non-gluten proteins. Analysis of the salt-soluble proteins from a transgenic line in which omega-1,2 gliadins were eliminated by RNA interference indicated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. Conclusions: In this study, we constructed a proteomic map of the non-gluten protein fraction of wheat flour from the US wheat Butte 86 that complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining which wheat proteins that should be targeted in future gene editing experiments to reduce the immunogenic potential of wheat flour.

The alpha gliadins are a group of more than 20 proteins with very similar sequences that comprise about 15%–20% of the total flour protein and contribute to the functional properties of wheat flour dough. Some alpha gliadins also contain immunodominant epitopes that trigger celiac disease, a chronic autoimmune disease that affects approximately 1% of the worldwide population. In an attempt to reduce the immunogenic potential of wheat flour from the U.S. spring wheat cultivar Butte 86, RNA interference was used to silence a subset of alpha gliadin genes encoding proteins containing celiac disease epitopes. Two of the resulting transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry. Although the RNA interference construct was designed to target only some alpha gliadin genes, all alpha gliadins were effectively silenced in the transgenic plants. In addition, some off-target silencing of high molecular weight glutenin subunits was detected in both transgenic lines. Compensatory effects were not observed within other gluten protein classes. Reactivities of IgG and IgA antibodies from a cohort of patients with celiac disease toward proteins from the transgenic lines were reduced significantly relative to the nontransgenic line. Both mixing properties and SDS sedimentation volumes suggested a decrease in dough strength in the transgenic lines when compared to the control. The data suggest that it will be difficult to selectively silence specific genes within families as complex as the wheat alpha gliadins. Nonetheless, it may be possible to reduce the immunogenic potential of the flour and still retain many of the functional properties essential for the utilization of wheat.

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

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.

Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Improving end-use quality and disease resistance are important goals in wheat breeding. The genetic loci controlling these traits are highly complex, consisting of large families of prolamin and resistance genes with members present in all three homeologous A, B, and D genomes in hexaploid bread wheat. Here, orthologous regions harboring both prolamin and resistance gene loci were reconstructed and compared to understand gene duplication and evolution in different wheat genomes. Comparison of the two orthologous D regions from the hexaploid wheat Chinese Spring and the diploid progenitor Aegilops tauschii revealed their considerable difference due to the presence of five large structural variations with sizes ranging from 100 kb to 2 Mb. As a result, 44% of the Ae. tauschii and 71% of the Chinese Spring sequences in the analyzed regions, including 79 genes, are not shared. Gene rearrangement events, including differential gene duplication and deletion in the A, B, and D regions, have resulted in considerable erosion of gene collinearity in the analyzed regions, suggesting rapid evolution of prolamin and resistance gene families after the separation of the three wheat genomes. We hypothesize that this fast evolution is attributed to the co-evolution of the two gene families dispersed within a high recombination region. The identification of a full set of prolamin genes facilitated transcriptome profiling and revealed that the A genome contributes the least to prolamin expression because of its smaller number of expressed intact genes and their low expression levels, while the B and D genomes contribute similarly.

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differences in retention times (RTs) for subunits corresponding to HMW-GS alleles were determined using 16 standard wheat cultivars with known HMW-GS compositions. Subunits that were not identified unambiguously by RP-HPLC were distinguished by SDS-PAGE or inferred from association with linked subunits. The method was used to verify the allelic compositions of 32 Korean wheat cultivars previously determined using SDS-PAGE and to assess the compositions of six new Korean cultivars. Three cultivars contained subunits that were identified incorrectly in the earlier analysis. The improved RP-HPLC method combined with conventional SDS-PAGE provides for accurate, efficient and reliable identification of HMW-GS and will contribute to efforts to improve wheat end-use quality.