Total Glutenin Research Articles

Technological quality of field grown transgenic lines of commercial wheat cultivars expressing the 1Ax1 HMW glutenin subunit gene

Ten transgenic lines were studied which expressed a transgene encoding HMW subunit 1Ax1 in three elite spring wheat cultivars: Imp, Canon and Cadenza. These lines contained one to five copies of the transgene and the 1Ax1 subunit was expressed as 1–20% of the total glutenin protein. These lines were grown in field trials in a continental, arid climate (Martonvásár, Hungary) over two years (2004, 2005). The expression of the transgenes and their effects on the grain properties were stably inherited over the two years. Significant differences in yield were observed between three of the transgenic lines and the original genotypes, but no differences were found in their adaptiveness. Clear differences were found in the technological and rheological properties of four lines, with all the parameters characterising dough strength and extensibility (GI, W, G, Re, Ext, A) changing significantly. These differences were associated with increases in the ratio of HMW/LMW subunits and decreases in the ratios of 1Dx/1Dy and 1Bx/1By subunits. Two transgenic lines of cv Imp had high over-expression of the 1Ax1 subunit which in one line resulted in an overstrong type of dough, similar to that described previously for lines over-expressing HMW subunit 1Dx5. Transformation of cvs. Canon and Cadenza resulted in two lines with increased dough stability due to the significantly improved gluten quality. It is concluded that significant changes in the structure of the glutenin polymers caused by the altered ratio of x-type to y-type HMW subunits led to the changes in flour functional properties.

Influence of gluten proteins on technological properties of Croatian wheat cultivars

The gluten proteins of 15 winter wheat cultivars grown in eastern Croatia were studied for their contribution to the bread-making quality. Composition of high-molecular-weight glutenin subunits (HMW-GS) was analyzed by SDS-PAGE, while the quantity of gluten proteins was determined by combined extraction/RP-HPLC procedure. The results of the linear correlation analysis carried out on the particular gluten proteins and technological properties showed that the amount of total gluten content highly correlates with protein content. Among gluten proteins, the glutenins showed higher correlation with protein content, with pronounced influence of HMW-GS, than gliadins. Wet gluten content was significantly correlated to total gliadin quantity. Gluten index as gluten quality parameter was positively influenced by total glutenins and low-molecular-weight glutenin subunits (LMW-GS), and negatively, by the ratios of gliadin to glutenin (Gli/Glu), whereas the amount of gliadins was not important. Dough development time was strongly correlated with total gluten content, total glutenins and the Gli/Glu ratio. Dough mixing resistance was strongly affected by total glutenin content with pronounced influence of HMW-GS. Degree of dough softening is mainly negative influenced by total glutenins and ratio of Gli/Glu. Farinograph quality number as flour quality index was highly positively correlated with total glutenins, with emphasized influence of HMW-GS. The Gli/Glu ratio had the highest influence on dough maximum resistance. Dough extensibility showed moderate correlation with total gliadins. The results of the linear correlation indicated that loaves volumes were significantly influenced by total gluten proteins, HMW-GS and LMW-GS.

Evidence that Minor Sprout Damage Can Lead to Significant Reductions in Gluten Strength of Winter Wheats

ABSTRACTSeventeen winter wheat lines were grown in triplicate plots at Warsaw, Painter, and Blacksburg, VA, during the 1999–2000 growing season. Hagberg falling numbers, protein content, farinograms, sedimentation volumes, and total glutenin content were determined for flours derived from 153 harvested wheat samples. Over three inches (8.2 cm) of rain fell during the week before harvest in Blacksburg, resulting in falling numbers of 100 for Recital and 137 for Heyne, two severely sprouted Blacksburg wheat samples, and falling numbers <250 in three other Blacksburg wheats. There were no significant differences across locations in falling numbers of four wheat lines, and one line had significantly greater falling numbers in Blacksburg than in Warsaw or Painter. All 18 Blacksburg flours had a significantly higher mixing tolerance index (MTI) and shorter departure times (DT) than corresponding Warsaw flours. Weaker gluten strength of Blacksburg flours suggestss that all 18 Blacksburg wheats were sprout‐damaged and contained active proteases. However, according to falling number data, five of these Blacksburg wheats were sprout‐free with falling numbers >400. These data indicate that Hagberg falling number should not be used as the sole criteria for determining the degree of sprout damage in wheat because it does not quantify, nor always accurately reflect changes in protein composition and quality due to grain weathering.

Impact of the fungal protease produced by Fusarium culmorum on the protein quality and breadmaking properties of winter wheat

Samples artificially infected by Fusarium culmorum were analyzed to assess the effects of Fusarium infection on the protein quality of winter wheat (Triticum aestivum L.). The Fusarium infection did not noticeably influence either the crude protein content or the water absorption ability of the wheat flour. The protease activity found in the wheat flour was inversely correlated to the sedimentation value. In contrast, it was positively correlated to both the free amino acid content and the degree of infection as expressed in Fusarium protein equivalents (FPE) quantified by an enzyme-linked immunosorbent assay. A distinct reduction in the content of both total glutenin and high molecular weight glutenin subunits was detected in the seriously infected samples (FPE>20 µg g−1). The infection with Fusarium impaired the dough quality and led to a deformed loaf shape. The fungal protease was active over both a wide range of temperatures (from 10 to 100 °C) and a wide range of pH values (from 4.5 to 8.5). The maximum protease activity was displayed at 50 °C in the pH range 6.0–8.0. This property indicates that the protease produced by F. culmorum may impair storage proteins throughout all the processing procedures associated with wheat flour, thereby causing weak dough properties and, consequently, unsatisfactory bread quality.

Relationship Between the Qualitative and Quantitative Compositions of Gluten Protein Types and Technological Properties of Synthetic Hexaploid Wheat Derived from Triticum durum and Aegilops tauschii

ABSTRACTThe contribution of the diploid wheat species Aegilops tauschii (Coss.) Schmall to the technological properties of bread wheat (Triticum aestivum L.) was previously studied by the investigation of synthetic hexaploids derived from tetraploid durum wheat (T. turgidum L.) and three diploid Ae. tauschii lines. The results indicated that bread volume, gluten index, SDS‐sedimentation volume, and maximum resistance of gluten were significantly influenced by the Ae. tauschii lines. To determine the relationship between technological properties and qualitative and quantitative compositions of gluten proteins, the flours of parental and synthetic lines were extracted using a modified Osborne fractionation. Gliadin and glutenin fractions were then characterized by reversed‐phase (RP) HPLC on C8 silica gel. The HPLC patterns revealed typical differences between synthetic and parental lines. The gliadin patterns of three synthetic lines and the glutenin patterns of two synthetic lines were more similar to that of the diploid Ae. tauschii parents involved in the hybrids. In the glutenin pattern of one synthetic line, characteristics from both Ae. tauschii and the durum wheat parents were observed. The amount of total gliadin and gliadin types of the synthetic lines was mostly intermediate between those of the durum and Ae. tauschii parents. The amounts of total glutenin and glutenin types (HMW and LMW subunits) of the synthetic lines were generally higher than those of the parental lines, and the ratio of gliadins to glutenins was significantly decreased. High positive correlations were found between the amount of total glutenins, HMW, and LMW subunits and bread volume, maximum resistance and extension area of gluten, and SDS‐sedimentation volume. The ratio of gliadins to glutenin subunits had a strong negative influence on these properties. The protein content of the flours and the amount of total gluten proteins were not correlated with any of the technological properties. Results on the relationship between biochemical characteristics and the breadmaking properties indicated that wheat prebreeding would benefit from studies on protein types and quantification in the choice of parents. In addition, the potential of the diploid Ae. tauschii for improvement of breadmaking quality should be further exploited.

Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale

Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum.

Comparative investigations of gluten proteins from different wheat species

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid). The flours were extracted to yield total endosperm proteins and the gluten protein fractions (gliadins and glutenin subunits). The extracts were characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase HPLC; both methods revealed that gluten protein groups and types known from common wheat (ω-, α-, γ-gliadins, HMW and LMW subunits of glutenin) were present in all species. The HPLC platterns of gliadins and glutenin subunits from species with the same genome composition (common wheat/spelt or durum wheat/emmer) were related, and those of einkorn quite different. According to the quantities determined by reversed-phase HPLC, α-gliadins were predominant in most cases, followed by γ-gliadins and LMW subunits; ω-gliadins and HMW subunits were generally minor components. Common wheats were characterised by the highest proportions of total glutenins and HMW subunits, which are known to be important for breadmaking quality. Moreover, the lower ratio of gliadins to glutenins was typical. Emmer had the lowest proportions of total glutenins and of HMW and LMW subunits, together with einkorn the highest proportion of α-gliadins, and, by far, the highest ratio of gliadins to glutenins. The values for spelt and durum wheat were mostly in a medium range between common wheats, emmer, and einkorn, respectively. Amongst common wheats, spring wheat was characterised by more balanced quantities of α- and γ-gliadins, and wheat rye hybrid by the highest proportions of ω-gliadins.

Environmental effects on the quality of two wheat genotypes: 1. quantitative and qualitative variation of storage proteins

A new method of sequential extraction of proteins followed by quantitative and qualitative determination by reverse-phased high-performance liquid chromatography (RP-HPLC) was used to analyse the effect of environmental conditions (3N fertilisation rates, two varieties, two sites and two growing seasons) on quantitative and qualitative variation of wheat storage proteins. The results showed that N supply (fertilisation and site) was the most important environmental factor affecting protein content and composition. The most important effect was quantitative: the total protein, protein unit and subunit contents increased with the supply of nitrogen to the grain. As grain protein increased, the gliadin and glutenin contents and the gliadin to glutenin ratio increased. Gliadin showed a higher correlation with total protein content than glutenin. In the variety Rinconada, glutenin content was higher than that in Bancal. As total glutenin increased, both high and low-molecular weight (HMW and LMW) fractions increased and their ratio (HMW/LMW) did not change significantly, despite a slope of LMW subunits two times greater (0.69) than that of HMW fractions (0.31). The quantity of each HMW subunit (RP-HPLC peak) increased with total HMW-glutenin, but their relative percentage increased for some peaks and decreased for others. The composition of the HMW subunit was more stable in Bancal than in Rinconada. This general pattern of variation was also characteristic of the LMW subunit. Concerning gliadin composition we noted that the content of different gliadin monomers (RP-HPLC peaks) or pools of monomers (α-, β-, γ-, ω-gliadin) increased with total gliadin content. The proportion of certain peaks was stable whereas the contribution of other peaks was related to the variation in gliadin content and depended on the variety.

Overexpression of a Gluten Protein in Transgenic Wheat Results in Greatly Increased Dough Strength

The transgenic wheat line B73-6-1 contains additional genes encoding a gluten protein called HMW subunit 1Dx5, resulting in a four-fold increase in the proportion of this component in the seed proteins and corresponding increases in the proportions of total HMW subunits and total glutenins. This is associated with a dramatic increase in dough strength, as measured using a small scale Mixograph, with dough produced from B73-6-1 being too strong to be used in conventional breadmaking.

Effects of Wheat Bug (Eurygaster maura) Protease on Glutenin Proteins

ABSTRACTProteolytic degradation of 50% 1‐propanol insoluble (50PI) glutenin of six common wheat cultivars by wheat bug (Eurygaster maura) protease was investigated using reversed‐phase HPLC. Wheat at the milk‐ripe stage was manually infested with adult bugs. After harvest, bug‐damaged kernels were blended (2:1, kernel basis) with undamaged grain of the same cultivar. Samples of ground wheat were incubated in distilled water for different times (0, 30, 60, and 120 min). The incubated whole meal samples were subsequently freeze‐dried and stored until analysis. The degree of proteolytic degradation of 50PI glutenin was determined based on the quantity of total glutenin subunits (GS), high molecular weight GS (HMW‐GS), and low molecular weight GS (LMW‐GS). For ground wheat samples incubated for ≥30 min, 50PI glutenin was substantially degraded as evidenced by a >80% decrease on average in total GS, HMW‐GS, and LMW‐GS. Some cultivars showed different patterns of glutenin proteolysis as revealed by differences in the ratios of HMW‐GS to LMW‐GS between sound and bug‐damaged samples; a significant decrease in this ratio was found for four cultivars. This evidence, combined with other observations, indicated that there were intercultivar differences in polymeric glutenin resistance to the protease of the wheat bug Eurygaster maura. While the nature of this resistance is unknown, it should be possible to select and develop wheat cultivars with improved tolerance for wheat bug damage. Propanol insoluble glutenin, which corresponds to relatively large glutenin polymers, appears to be an excellent quantitative marker for this purpose.

Characterization of the Carbohydrates of Nonreduced Glutenin Fractionated by Multistacking SDS‐PAGE from Two Hard Red Spring Wheat Flours

ABSTRACTNative (nonreduced) glutenin aggregates of two hard red spring wheat flours (Len of good quality and line 205 of poor quality) containing the same high molecular weight glutenin subunits (2*, 7+9, and 5+10) were investigated for the possibility of glycosylation. Glutenins isolated by pH‐precipitation were separated and purified under nonreducing conditions into five different molecular weight species by multistacking SDS‐PAGE and by transfer to polyvinylidene difluoride (PVDF) membranes by electroblotting. Carbohydrate compositions of the total glutenin fraction and of the different molecular weight glutenin species separated on the stacking gels (4–12% acrylamide) were determined. More total carbohydrates were found in the total glutenin of the line 205 flour (2.33%) than in that of the Len flour (1.87%), with glucose and xylose contributing to the greater total amount in line 205. However, the total glutenin of Len had approximately twice as much of the total of arabinose, mannose, and galactose as line 205 had. After purification by electrophoresis, smaller amounts of monosaccharides (glucose, xylose, arabinose, galactose, and mannose) in the different molecular species were detected. After electroblotting to PVDF membrane to increase purification, no arabinoxylans were found. Following extraction of glutenin from membranes, the β‐elimination procedure in mild base under reducing conditions was used. In that procedure, the mono‐ or oligosaccharide side chains are released from the protein core, and the sugars originally involved in the protein‐sugar linkage are reduced to the sugar alcohol. After derivatization (with trimethylsilyl), samples were analyzed by gas chromatography and mass spectrometry. Glucose, galactose, and a small amount of mannitol were found in most of the different glutenin aggregates, except that mannitol was not found in the 8% stacking gel glutenin fraction. The content of mannitol was greater in the higher molecular weight glutenin species at the 4 and 6% origins. These results support the hypothesis that the carbohydrates and protein in the glutenin macropolymer may be covalently linked.

Identification of the major water/salt insoluble wheat proteins involved in cereal hypersensitivity

Several studies have investigated water/salt soluble proteins which comprise 50% of the proteins in wheat. The remaining 50% of wheat proteins, are water/salt insoluble proteins of which there is limited information on their role in cereal hypersensitivity. To investigate the allergenicity of the water/salt insoluble gliadin and glutenin proteins (prolamins). RAST, electrophoresis and Western blotting were used to identify water/salt insoluble wheat allergens. Competitive RAST inhibition was conducted to investigate cross-reactivity between prolamins and water/salt soluble wheat proteins. Specific IgE to alpha-gliadin and to total glutenins were detected in all sera. IgE to beta-, gamma-, fast omega-, and slow omega-gliadin were present in lower numbers of sera. Prolamin allergens of 90-11 kDa were identified by immunoblotting. Water/salt soluble proteins crossreacted with alpha-gliadin and total glutenins. Individuals who are hypersensitive to water/salt soluble wheat proteins produce specific IgE to water/salt insoluble wheat proteins. Western blotting has shown that gliadins, glutenins and proteins with similar molecular weights as the endogenous water/salt soluble wheat enzyme inhibitors are important allergens. Alpha and fast omega- are the most allergenic gliadins. The water/salt insoluble proteins share cross-reacting epitopes with water/salt soluble proteins. These data show that the numbers of proteins involved in the development of cereal hypersensitivity is greater than previously believed and that the development of specific IgE to alpha-gliadin may in part depend on the presence of cross-reacting antibodies to water/salt soluble flour allergens.

Indian Wheat Cultivars: Correlation between Quality of Gluten Proteins, Rheological Characteristics of Dough and Tandoori Roti Quality

Physico-chemical, rheological characteristics and quality of tandoori roti were determined for whole wheat flours from eight commercial wheat cultivars grown at one location. Tandoori roties baked from these flours indicated wide differences in baking performance. Among the varieties, Cpan-3004, GW-180 and K-8804 and WL-1562 were found suitable for the preparation of tandoori roti. These four varieties had higher water absorption capacity (718–735 ml kg-1) when measured in a farinograph at an optimum tandoori dough consistency of 400 BU. The doughs from these varieties also had higher cohesiveness (0·38–0·45) and extensibility (114–121 mm) than other varieties. Total protein, gluten as well as total glutenin (soluble and insoluble) contents were found to be maximum in these four varieties. Varieties having Glu-1 score of 6 were found to be more suitable for roti preparation. The prediction equation derived for correlating total protein and total glutenin in flour with overall sensory score of roti was found highly significant (R2 = 0·92). The results suggest that the medium hard wheats are suitable for the preparation of tandoori roti. © 1997 SCI.

Indian Wheat Cultivars: Correlation between Quality of Gluten Proteins, Rheological Characteristics of Dough and Tandoori Roti Quality

Physico-chemical, rheological characteristics and quality of tandoori roti were determined for whole wheat flours from eight commercial wheat cultivars grown at one location. Tandoori roties baked from these flours indicated wide differences in baking performance. Among the varieties, Cpan-3004, GW-180 and K-8804 and WL-1562 were found suitable for the preparation of tandoori roti. These four varieties had higher water absorption capacity (718–735 ml kg-1) when measured in a farinograph at an optimum tandoori dough consistency of 400 BU. The doughs from these varieties also had higher cohesiveness (0·38–0·45) and extensibility (114–121 mm) than other varieties. Total protein, gluten as well as total glutenin (soluble and insoluble) contents were found to be maximum in these four varieties. Varieties having Glu-1 score of 6 were found to be more suitable for roti preparation. The prediction equation derived for correlating total protein and total glutenin in flour with overall sensory score of roti was found highly significant (R2 = 0·92). The results suggest that the medium hard wheats are suitable for the preparation of tandoori roti. © 1997 SCI.

Characterization and Quantification of Native Glutenin Aggregates by Multistacking Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE) Procedures

ABSTRACTNative glutenin aggregates of two different quality flours containing the same high molecular weight (HMW) glutenin subunit compositions were investigated by multistacking sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) procedures. Five stacking gels (4, 6, 8, 10, and 12%) with a separating (resolving) gel of 14% were used to separate nonreduced glutenin aggregates solubilized from flour by SDS sodium phosphate buffer. There were large differences in protein solubility of the two flours. It took 8 hr to extract 91% of total proteins from the strong flour (variety Len) while it took only 2 hr for the weak flour sample 205. Total glutenin proteins and the proportions of glutenins at the different origins (including the origin of the 14% separating gel) were quantified by high‐resolution densitometry procedures. As the duration of extraction increased, both total glutenins and glutenins at the 4% origins increased. The good quality flour Len had a higher total glutenin protein (3% more) and higher proportion of glutenins with the largest molecular sizes (also 3% more) at the 4% origins than the poor quality flour sample 205. After glutenin aggregates from each origin were reduced and analyzed by SDS‐PAGE, the largest glutenins at the 4% origin contained twice the amount of total HMW glutenin subunits when compared to the smaller aggregates at the 12% origin. Among the total HMW glutenin subunits, the proportion of subunit 5 (which published literature reports to be the largest molecular weight based on calculations of DNA‐derived amino acid sequence analysis) was twice that at the 12% origin. A randomized structure of native glutenins is proposed based on the results of our investigations.

Production of monoclonal antibodies to gluten proteins and their use in developing tests for gluten quality

There is a need for rapid, simple tests to give added assurance in gluten quality evaluation which address the needs of plant breeders and the milling and baking industry. As part of fundamental research into gluten protein structure‐function relationships, a library of monoclonal antibodies (MAbs) has been developed to various gluten protein fractions, including both high and low molecular weight subunits of glutenin. The difficulties presented by working with insoluble, heterogeneous gluten proteins in raising MAbs of the requisite specificity are discussed. Several of the MAbs have been used to develop rapid immunoassays which are capable of analyzing a single sample in 10 min, and these have been applied to the quantification of total glutenin proteins, and high and low molecular weight subunits of glutenin in flour samples, illustrating the potential of immunotechnology for monitoring flour quality.

Monoclonal antibodies that recognize the repeat motif of the S-poor prolamins

A panel of murine monoclonal antibodies (MAbs) was generated to the total glutenin fraction of wheat. Two of the MAbs (IFRN 0061 and 00614) were characterized in terms of their binding to cereal prolamins using immunoassay and immunoblotting techniques. Their specificities, as determined by immunoassay, were restricted, binding mainly to the S-poor prolamins of barley, wheat and rye, and they exhibited a high affinity for the major repeat motif, PQQPFPQQ, present in these polypeptides. Analysis of genetic stocks by immunoblotting showed that the epitopes recognized by these MAbs are present in the polymeric LMW subunits of glutenin encoded by genes on the short arms of the group 1 chromosomes of wheat.

Dough and baking quality of wheat lines deficient in glutenin subunits controlled by the Glu-A1, Glu-B1 and Glu-D1 loci

This study evaluated the quality potential of seven synthetic hexaploid wheats (2n=6x=42, AABBDD) expressing only allelic variation at Glu-D1 of Aegilops tauschii (SHWSD). Major quality parameters related to dough strength, gluten proteins (including high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS), gliadins), and their ratios between SHWSD and the weak gluten wheat control Chuannong 16 (CN16) were measured in at least three environments (except STD7). The zeleny sedimentation value (ZSV), dough development time (DDT), dough stability time (DST), and farinograph quality number (FQN) of SHWSD were considered stable under different environments, with their respective ranges being 8.00–17.67 mL, 0.57–1.50 min, 0.73–1.80 min, and 9.50–27.00. The ZSV, DDT, DST, and FQN of SHWSD were smaller than those of CN16, suggesting that SHWSD had a weaker dough strength than CN16. Although SHWSD had a lower gluten index than CN16, its wet and dry gluten contents were similar to or even higher than those of CN16 in all environments tested. The protein content of grains (12.81–18.21%) and flours (14.20–20.31%) in SHWSD was higher than that in CN16. The amount of HMW-GS in SHWSD sharply decreased under the expression of fewer HMW-GS genes, and the LMW-GS, gliadins, and total glutenins were simultaneously increased in SHWSD in comparison with CN16. Moreover, SHWSD had higher ratios of LMW-GS/glutenin and gliadin/glutenin but a lower ratio of HMW-GS/glutenin than CN16. These results provide necessary information for the utilization of SHWSD in weak-gluten wheat breeding.

Chromosomal control of glutenin subunits in aneuploid lines of wheat: analysis by reversed-phase high-performance liquid chromatography

Glutenin subunits from nullisomic-tetrasomic and ditelocentric lines of the hexaploid wheat variety 'Chinese Spring' (CS) and from substitution lines of the durum wheat variety 'Langdon' were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) at 70 °C using a gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid. Nineteen subunits were detected in CS. The presence and amounts of four early-eluted subunits were found, through aneuploid analysis, to be controlled by the long arms of chromosomes 1D (1DL) (peaks 1-2) and 1B (1BL) (peaks 3-4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that these four subunits are the high molecular weight subunits of glutenin, which elute in the order 1Dy, 1Dx, 1By, and 1Bx. Similar amounts of 1DL subunits were present (6.3 and 8.8% of total glutenin), but 1BL subunits differed more in abundance (5.4 and 9.5%, respectively). Results indicate that most late-eluting CS glutenin subunits were coded by structural genes on the short arms of homoeologous group 1 chromosomes: 6 by 1DS, 5 by 1AS, and 4 by 1BS. Glutenin of tetraploid 'Langdon' durum wheat separated into nine major subunits: 6 were coded by genes on 1B chromosomes, and 3 on 1A chromosomes. Gene locations for glutenin subunits in the tetraploid durum varieties 'Edmore' and 'Kharkovskaya-5' are also given. These results should make RP-HPLC a powerful tool for qualitative and quantitative genetic studies of wheat glutenin.