A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 ≈ 3 > 1 > 6 ≈ 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SDS-PAGE. The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order.     

Effect of temperature, time and wheat gluten moisture content on wheat gluten network formation during thermomolding

A plastic-like material can be obtained by thermomolding wheat gluten protein which consists of glutenin and gliadin. We studied the effect of molding temperature (130-170 °C), molding time (5-25 min) and initial wheat gluten moisture content (5.6-18.0%) on the gluten network. Almost no glutenins were extractable after thermomolding irrespective of the molding conditions. At the lowest molding temperature, the extractable gliadin content decreased with increasing molding times and moisture contents. This effect was more pronounced for the α- and γ-gliadins than for the ω-gliadins. Protein extractabilities under reducing conditions revealed that, at this molding temperature, the cross-linking was predominantly based on disulfide bonds. At higher molding temperatures, also non-disulfide bonds contributed to the gluten network. Decreasing cystine contents and increasing free sulfhydryl and dehydroalanine (DHA) contents with increasing molding temperatures and times revealed the occurrence of β-elimination reactions during thermomolding. Under the experimental conditions, the DHA derived cross-link lanthionine (LAN) was detected in all gluten samples thermomolded at 150 and 170 °C. LAN was also formed at 130 °C for gluten samples containing 18.0% moisture. Degradation was observed at 150 °C for samples thermomolded from gluten with 18.0% moisture content or thermomolded at 170 °C for all moisture contents.     

Improved control of lettuce drop caused by Sclerotinia sclerotiorum using Contans combined with lignin or a reduced fungicide application

The use of Contans (Coniothyrium minitans) in Belgian commercial lettuce greenhouses has so far not always fulfilled the expectations of growers. With the aim of improving the efficacy of control of lettuce drop (Sclerotinia sclerotiorum), Contans applied alone and Contans combined with Radix (Trichoderma asperellum and Trichoderma gamsii), Kraft pine lignin and Rovral was compared in three successive lettuce crops in a naturally infested greenhouse. At harvest of each crop, the effect on disease incidence, and the effect on viability and fungal infection of sclerotia buried prior to each crop was evaluated. Contans applied alone negatively affected the sclerotial viability, but did not reduce lettuce drop symptoms. The combination of Contans with Kraft pine lignin, on the other hand, reduced sclerotial viability and lettuce drop compared with the untreated control in the last crop. Furthermore, when Contans was combined with Kraft pine lignin, a higher number of infected sclerotia was observed compared with Contans applied alone. When Radix was added to the combination, there was no extra benefit for the control of lettuce drop. The combination of one Rovral application and Contans resulted in a significant control of lettuce drop together with a negative effect on sclerotial survival. Two Rovral applications, by contrast, reduced the ability of C. minitans to parasitize and kill the sclerotia. To conclude, integration of Contans with a reduced Rovral application and with Kraft pine lignin has potential to improve the control of lettuce drop in commercial greenhouses.     

Characterisation of the first wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase resembling mammalian counterparts

A formerly unknown nucleotide pyrophosphatase/phosphodiesterase (NPP) of wheat (Triticum aestivum L.) (TaNPP) was recombinantly produced in Pichia pastoris (TaNPPr) following assembly of the sequence out of wheat ESTs using sequences of known NPPs. Simultaneously, a phosphodiesterase was purified to homogeneity from wheat germs, characterised and identified as TaNPP. TaNPP contains the highly conserved catalytic substrate and metal binding residues and displays properties [high pH optimum, glycosylation, high thermostability and inhibition by EDTA and adenosine 5′-monophosphate (AMP)] similar to those of mammalian enzymes characterised earlier. The sequence of TaNPP includes that of a putative transmembrane domain, a characteristic of most NPPs. Both recombinant and native TaNPP partially occur as oligomeric proteins on SDS PAGE. Upon addition of DTT, both migrate as monomeric proteins. Part of the native wheat protein even occurs in its truncated form, thus lacking the transmembrane region. Out of a range of tested natural substrates, TaNPP had the highest affinity towards adenine containing nucleotides. While Michaelis-Menten kinetics were valid in the low substrate concentration range, at higher concentrations, they were no longer applicable. TaNPP shows no similarity to the recently characterised rice and barley enzymes and, is hence, one of the first characterised plant NPPs resembling mammalian NPPs.     

β-Elimination reactions and formation of covalent cross-links in gliadin during heating at alkaline pH

The aim of this study was to increase insight into gluten polymerisation. While previous research on this topic focused on disulfide (SS) bonds, the present paper focuses on cross-links based on dehydro-protein formation through β-elimination reactions. Gliadin, the monomeric fraction of gluten containing no free sulfhydryl (SH) groups, was heated for 120 min at pH 8.0 and 130 °C, and cross-link formation was evaluated by determining extractability in sodium dodecyl sulfate containing buffer, reaction products of β-elimination reactions, and cross-links involving the latter. Heating decreased gliadin extractability. Reduction of SS bonds increased extractability of heated gliadin, but did not restore it to that of non-heated gliadin, suggesting contribution of both SS and non-SS bonds to gliadin cross-linking. Decreased SS levels and the presence of dehydroalanine and SH groups in heated gliadin samples indicated cleavage of SS bonds by β-elimination reactions. Some of the formed free SH groups were then involved in oxidation and/or SH–SS interchange reactions leading to intermolecular SS bonds. In addition, amino acid analysis revealed formation of an irreversible non-SS cross-link between dehydroalanine and the free SH group of cysteine, namely lanthionine. In conclusion, non-SS bonds may well contribute to the gluten network under specified reaction conditions.     

Assignments of proton populations in dough and bread using NMR relaxometry of starch, gluten, and flour model systems

Starch-water, gluten-water, and flour-water model systems as well as straight-dough bread were investigated with (1)H NMR relaxometry using free induction decay and Carr-Purcell-Meiboom-Gill pulse sequences. Depending on the degree of interaction between polymers and water, different proton populations could be distinguished. The starch protons in the starch-water model gain mobility owing to amylopectin crystal melting, granule swelling, and amylose leaching, whereas water protons lose mobility due to increased interaction with starch polymers. Heating of the gluten-water sample induces no pronounced changes in proton distributions. Heating changes the proton distributions of the flour-water and starch-water models in a similar way, implying that the changes are primarily attributable to starch gelatinization. Proton distributions of the heated flour-water model system and those of fresh bread crumb are very similar. This allows identifying the different proton populations in bread on the basis of the results from the model systems.     

Identification of isopeptide bonds in heat-treated wheat gluten peptides

Results in this paper confirm heat-induced isopeptide bond formation in wheat gluten. Heating (24 h, 130 °C) of wheat gluten [moisture content 7.4%] decreased its extractability in sodium dodecyl sulfate containing buffer (pH 6.8), even after reduction of disulfide (SS) bonds. Thus, both SS bonds and non-SS bonds were responsible for the extractability loss. Cross-links of the lysinoalanine and lanthionine type were not present in the heated samples, but heat treatment reduced levels of available amino groups. Heating of purified and alkylated high molecular weight glutenin subunits (HMW-GS) under similar conditions also resulted in extractability loss, demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine. These observations indicated that heat treatment had induced isopeptide bond formation, resulting in larger and unextractable molecules. Heating HMW-GS lysine- and glutamine-containing peptides induced the formation of isopeptide bonds, thereby supporting the above hypothesis. The level of isopeptide bond formation increased with heating time.