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Key Message: The suppression of the HYD-1 gene by a TILLING approach increases the amount of β-carotene in durum wheat kernel. Vitamin A deficiency is a major public health problem that affects numerous countries in the world. As humans are not able to synthesize vitamin A, it must be daily assimilated along with other micro- and macronutrients through the diet. Durum wheat is an important crop for Mediterranean countries and provides a discrete amount of nutrients, such as carbohydrates and proteins, but it is deficient in some essential micronutrients, including provitamin A. In the present work, a targeting induced local lesions in genomes strategy has been undertaken to obtain durum wheat genotypes biofortified in provitamin A. In detail, we focused on the suppression of the β-carotene hydroxylase 1 (HYD1) genes, encoding enzymes involved in the redirection of β-carotene toward the synthesis of the downstream xanthophylls (neoxanthin, violaxanthin and zeaxanthin). Expression analysis of genes involved in carotenoid biosynthesis revealed a reduction of the abundance of HYD1 transcripts greater than 50% in mutant grain compared to the control. The biochemical profiling of carotenoid in the wheat mutant genotypes highlighted a significant increase of more than 70% of β-carotene compared to the wild-type sibling lines, with no change in lutein, α-carotene and zeaxanthin content. This study sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat and provides new genotypes that represent a good genetic resource for future breeding programs focused on the provitamin A biofortification through non-transgenic approaches., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Two expressed alleles of the 1Ay high-molecular-weight glutenin subunit (HMW-GS), 1Ay21* and 1AyT1 , previously introduced in durum and bread wheat, were separately introgressed into the Australian bread wheat ( Triticum aestivum L.) cv. Livingston. The developed lines had different allelic compositions compared to that of the parental cultivar ( 1Ax1 ), having either 1Ax21 + 1Ay21* or 1Ax1 + 1AyT1 at the Glu-A1 locus. Since 1Ax21 and 1Ax1 are known to have the same effects on quality, differences observed between the two sets of the developed lines are attributed to the two introgressed Ay genes. Yield and agronomic performance of the lines were evaluated in the field, and the protein, dough, and baking quality attributes were evaluated by large-scale quality testing. Results demonstrated that the subunit 1Ay21* increased unextractable polymeric protein by up to 14.3% and improved bread loaf volume by up to 9.2%. On the other hand, subunit 1AyT1 increased total grain protein by up to 9% along with dough elasticity. Furthermore, milling extraction was higher, and flour ash was lower in the 1Ay21* lines compared to the lines integrating 1AyT1 . Both sets of the 1Ay introgression lines reduced dough-mixing time compared to the recurrent parent Livingston. The results also showed that 1Ay21* had a higher potential to improve the baking quality than 1AyT1 under the Livingston genetic background. Both alleles showed the potential to be utilized in breeding programs to improve the breadmaking quality.

A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line were crossed and backrossed four times. At each step electrophoretic selection for the seeds that possessed, simultaneously, the complete set of high molecular weight glutenin subunits of N11 and the two high molecular weight secalins of rye, present in the 2D(2R) line, was carried out. Molecular cytogenetic analyses of the BC 4 F 8 generation revealed that the selection carried out produced a disomic addition line (2n = 44). The pair of additional chromosomes consisted of the long arm of chromosome 1R (1RL) from rye fused with the satellite body of the wheat chromosome 6B. Rheological analyses revealed that the dough obtained by the new addition line had higher quality characteristics when compared with the two parents. The role of the two additional high molecular weight secalins, present in the disomic addition line, in influencing improved dough characteristics is discussed.

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker's asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3 , CM16 and 0.28 . We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Drought stress is becoming more prevalent with global warming, and has been shown tohave large effects on gluten proteins linked to wheat bread making quality. Likewise, lowtemperature stress can detrimentally affect proteins in wheat. This study was done to determine thedifferential abundance of high molecular weight (HMW) glutenin proteins in a drought and lowtemperature stressed high quality hard red spring wheat cultivar (PAN3478), against a control. Thetreatments were applied in the greenhouse at the soft dough stage. HMW glutenin proteins wereextracted from the flour, and were separated by using two-dimensional gel electrophoresis. Proteinspots that had p values lower than 0.05 and fold values equal to or greater than 1.2 were consideredto be significantly differentially abundant. These proteins were further analyzed by using tandemmass 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 matchedeither HMW or low molecular weight (LMW) glutenin subunits. In the latter case, the C subunits ofLMW glutenins were notably found to be up-regulated under both stress conditions. All the proteinsthat have been identified can directly influence dough characteristics. Data are available viaProteomeXchange with the identifier PXD017578.

Macro- and micronutrients, essential for the maintenance of human metabolism, are assimilated daily through the diet. Wheat and other major cereals are a good source of nutrients, such as carbohydrates and proteins, but cannot supply a sufficient amount of essential micronutrients, including provitamin A. As vitamin A deficiency (VAD) leads to several serious diseases throughout the world, the biofortification of a major staple crop, such as wheat, represents an effective way to preserve human health in developing countries. In the present work, a key enzyme involved in the branch of carotenoids pathway producing β-carotene, lycopene epsilon cyclase, has been targeted by a Targeting Induced Local Lesions in Genomes (TILLING) approach in a "block strategy" perspective. The null mutant genotype showed a strong reduction in the expression of the lcyE gene and also interesting pleiotropic effects on an enzyme (β-ring hydroxylase) acting downstream in the pathway. Biochemical profiling of carotenoids in the wheat mutant lines showed an increase of roughly 75% in β-carotene in the grains of the complete mutant line compared with the control. In conclusion, we describe here the production and characterization of a new wheat line biofortified with provitamin A obtained through a nontransgenic approach, which also sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat.

Key Message: Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.

Background: Among wheat gluten proteins, the α-type gliadins are the major responsible for celiac disease, an autoimmune disorder that affects about 1% of the world population. In fact, these proteins contain several toxic and immunogenic epitopes that trigger the onset of the disease. The α-type gliadins are a multigene family, encoded by genes located at the complex Gli-2 loci., Results: Here, three bread wheat deletion lines (Gli-A2, Gli-D2 and Gli-A2/Gli-D2) at the Gli-2 loci were generated by the introgression in the bread wheat cultivar Pegaso of natural mutations, detected in different bread wheat cultivars. The molecular characterization of these lines allowed the isolation of 49 unique expressed genes coding α-type gliadins, that were assigned to each of the three Gli-2 loci. The number and the amount of α-type gliadin transcripts were drastically reduced in the deletion lines. In particular, the line Gli-A2/Gli-D2 contained only 12 active α-type gliadin genes (-75.6% respect to the cv. Pegaso) and a minor level of transcripts (-80% compared to cv. Pegaso). Compensatory pleiotropic effects were observed in the two other classes of gliadins (ω- and γ-gliadins) either at gene expression or protein levels. Although the comparative analysis of the deduced amino acid sequences highlighted the typical structural features of α-type gliadin proteins, substantial differences were displayed among the 49 proteins for the presence of toxic and immunogenic epitopes., Conclusion: The deletion line Gli-A2/Gli-D2 did not contain the 33-mer peptide, one of the major epitopes triggering the celiac disease, representing an interesting material to develop less "toxic" wheat varieties.

Gluten proteins are major determinants of the bread making quality of wheat, but also of important wheat-related disorders, including coeliac disease (CD), and allergies. We carried out a proteomic study using the total grain proteins from two low-gliadin wheat lines, obtained by RNAi, and the untransformed wild type as reference. The impact of silencing on both target and on non-target proteins was evaluated. Because of the great protein complexity, we performed separate analyses of four kernel protein fractions: gliadins and glutenin subunits, and metabolic and CM-like proteins, by using a classical 2D electrophoresis gel based approach followed by RP-HPLC/nESI-MS/MS. As a result of the strong down-regulation of gliadins, the HMW-GS, metabolic and chloroform/methanol soluble proteins were over-accumulated in the transgenic lines, especially in the line D793, which showed the highest silencing of gliadins. Basing on these data, and considering that metabolic proteins and chloroform/methanol soluble proteins (CM-like), such as the α-amylase/trypsin inhibitor family, β-amylase and serpins, were related to wheat allergens, further in vivo analysis will be needed, especially those related to clinical trials in controlled patients, to determine if these lines could be used for food preparation for celiac or other gluten intolerant groups., Biological Significance: Several enteropathies and allergies are related to wheat proteins. Biotechnological techniques such as genetic transformation and RNA interference have allowed the silencing of gliadin genes, providing lines with very low gliadin content in the grains. We report a proteomic-based approach to characterize two low-gliadin transgenic wheat lines obtained by RNAi technology. These lines harbor the same silencing fragment, but driven by two different endosperm specific promoters (γ-gliadin and D-hordein). The comprehensive proteome analysis of these transgenic lines, by combining two-dimensional electrophoresis and RP-HPLC/nESI-MS/MS, provided a large number of spots differentially expressed between the control and the transgenic lines. Hence, the results of this study will facilitate further safety evaluation of these transgenic lines., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Wheat high molecular weight glutenin subunit variation is important because of its great influence on glutenin polymer structure, that is related to dough technological properties. Among the different subunits, the pair Bx20 and By20 is known to have a negative effect on quality, but the reasons are not clear: Bx20 has two cysteines, which theoretically make this subunit a chain extender of the glutenin polymer, just like the other Bx subunits, showing four cysteines, two of which should be involved in intra-molecular disulfide bonds. By20 has never been characterized so far at molecular level. Here we report the nucleotide sequences of Bx20 and By20 genes isolated from the durum wheat cultivar 'Lira 45' and the validation of the corresponding deduced amino acid sequences by using MALDI-TOF and LC-MS/MS. Four nucleotide differences were identified in the Bx20 gene with respect to the deduced sequence present in NCBI, causing two amino acid substitutions. For the By20 subunit, nucleotide and amino acid sequences revealed a great similarity to By15, both at gene and protein levels, showing five nucleotide changes generating two amino acid differences. No evidence of post-translational modifications has been found. Hypotheses are formulated in regard to relationships with technological quality. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Environmental stress during grain filling may affect wheat protein composition, thus influencing its final quality. A proteomic approach was used to evaluate changes in storage protein composition under water stress of two Italian durum wheat (Triticum turgidum ssp. durum) cultivars, Ciccio and Svevo. The high-molecular-weight glutenin region increased progressively in both cultivars and under two water regimens. The L48-35 region, corresponding to low-molecular-weight (LMW) glutenin subunits, increased slightly during grain development and decreased under water stress in both cultivars. In particular, an s-type LMW related to superior technological quality was down-expressed in the early-mid period in Svevo and in the mid-late period in Ciccio. Finally, the L<35 region, corresponding to gliadin-like proteins, decreased slightly during grain development and increased under stress in both cultivars. Several α-gliadins, associated with immunological potential, increased their expression under water stress, especially in Svevo in the early-mid stage of grain filling.

Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

Background: Wheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase., Results: We performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing., Conclusions: Our results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera., Biological Significance: The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC-MS/MS analyses classical software and parameters were used as described in Material and methods. We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter., Results: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR)., Conclusion: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2-D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2- and 2.2-fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI-TOF and MALDI-TOF-TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress-related proteins. Many of the heat-induced polypeptides are considered to be allergenic for sensitive individuals.

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.

We carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that overexpresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding nontransformed genotype. Proteomic analyses showed that, during seed development, several classes of endosperm proteins were differentially accumulated in the transformed endosperm. As a result of the strong increase in the amount of the transgenic protein, the endogenous glutenin subunit, all subclasses of gliadins, and metabolic as well as chloroform/methanol soluble proteins were diminished in the transgenic genotype. The differential accumulation detected by proteomic analyses, both in mature and developing seeds, was paralleled by the corresponding changes in transcript levels detected by microarray experiments. Our results suggest that the most evident effect of the strong overexpression of the transgenic glutenin gene consists in a global compensatory response involving a significant decrease in the amounts of polypeptides belonging to the prolamin superfamily. It is likely that such compensation is a consequence of the diversion of amino acid reserves and translation machinery to the synthesis of the transgenic glutenin subunit.

Gliadins and glutenins are the main protein fractions present in wheat gluten. They are responsible for technological and nutritional quality of wheat based products. In particular, glutenins are mainly responsible for dough visco-elastic properties, whereas gliadins confer extensibility to dough and are the most important factor triggering celiac disease, the major human intolerance to gluten. Gliadins are monomeric proteins, whereas glutenins are polymers stabilized by disulfide bonds. Although they have distinctive structural characteristics, it is possible that some gliadins become part of the glutenin fraction because of mutations that affect cysteine number and distribution. Here, we provide evidence that a naturally mutated gamma-gliadin with an extra cysteine residue is incorporated into the polymeric fraction. This goal was achieved using an integrated approach involving heterologous expression, 2-DE, RP-HPLC and MS.

Low molecular weight glutenin subunits (LMW-GS) are typically subdivided into three groups, according to their molecular weights and isoelectric points, namely the B-, C-, and D groups. Enriched B- and C-type LMW-GS fractions extracted from the bread wheat cultivar Chinese Spring were characterized using high performance liquid chromatography (HPLC) directly interfaced with electrospray ionization mass spectrometry and HPLC coupled off-line with matrix-assisted laser desorption/ionization mass spectrometry, in order to ascertain the number and relative molecular masses of the components present in each fraction and determine the number of cysteine residues. About 70 components were detected in each of the fractions examined by the combined use of these two techniques, with 18 components common to both fractions. Analysis of the fractions after alkylation with 4-vinylpyridine allowed determination of the number of the cysteines present in about 40 subunits. The proteins detected were tentatively classified based on the relative molecular masses and number of cysteine residues. Cross-contamination was found in both B- and C- fractions, along with the presence of D-type LMW-GS. The two fractions also contained unexpected components, probably lipid transfer proteins and omega-gliadins. The presence of extensive microheterogeneity was suggested by the detection of several co-eluting proteins with minor differences in their molecular masses.

Wheat technological properties are correlated with the size of glutenin polymers, consisting of high and low molecular mass glutenin subunits, linked together by disulphide bonds. In order to unravel glutenin polymer structure, we considered three LMW-GS genes, which differ in the number of cysteine residues and in the repetitive domain length. The three LMW-GS genes have been expressed in Escherichia coli, and purified with a yield of 40-100 mg/l of culture volume, depending on protein type. Single polypeptides are being used in re-oxidation and micro-mixographic experiments, in order to detect the influence of the differential structural characteristics on glutenin polymer formation.

Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

Gli-D1-encoded omega gliadins of bread wheats show little variation; their electrophoretic patterns can be classified into two main groups which broadly resemble the patterns found in the cultivars Chinese Spring and in Cheyenne. B and D subunits of low molecular weight glutenin encoded by the chromosome 1D loci Glu-D3 and Gli-D1, respectively, also showed little variation. D subunits were found only in bread wheats with "Chinese Spring-type" omega gliadins and they all exhibited the same electrophoretic pattern. This material also showed very similar B subunits. "Cheyenne-type" bread wheats displayed the same electrophoretic distribution of chromosome 1D-encoded B subunits, although they were slightly different from that found in Cheyenne itself.