Cloning, expression and characterization of novel avenin-like genes in wheat and related species

B-Type avenin-like genes and proteins were characterized in 23 species of Triticeae. Southern blot analysis showed that the avenin-like genes belong to a multigene family. RT-PCR showed expression only in developing endosperms of wheat and related species, between 3 and 22 DPA (days post anthesis) with a peak between 11 and 15 DPA in wheat. The encoded proteins are cysteine-rich, containing 18–19 cysteine residues. An avenin-like protein from wheat was expressed in Escherichia coli, purified and used to raise polyclonal antibodies. These antibodies were used to detect b-type avenin-like proteins in endosperms of wheat and related species by western blotting.     

Thermoplastic films from wheat proteins

We show that the wheat proteins gluten, gliadin and glutenin can be compression molded into thermoplastic films with good tensile strength and water stability. Wheat gluten is inexpensive, abundantly available, derived from renewable resource and therefore widely studied for potential thermoplastic applications. However, previous reports on developing thermoplastics from wheat proteins have used high amounts of glycerol (30-40%) and low molding temperature (90-120 °C) resulting in thermoplastics with poor tensile properties and water stability making them unsuitable for most thermoplastic applications. In this research, we have developed thermoplastic films from wheat gluten, gliadin and glutenin using low glycerol concentration (15%) but high molding temperatures (100-150 °C). Our research shows that wheat protein films with good tensile strength (up to 6.7 MPa) and films that were stable in water can be obtained by choosing appropriate compression molding conditions. Among the wheat proteins, wheat gluten has high strength and elongation whereas glutenin with and without starch had high strength and modulus but relatively low elongation. Gliadin imparts good extensibility but decreased the water stability of gluten films. Gliadin films had strength of 2.2 MPa and good elongation of 46% but the films were unstable in water. Although the tensile properties of wheat protein films are inferior compared to synthetic thermoplastic films, the type of wheat proteins and compression molding conditions can be chosen to obtain wheat protein films with properties suitable for various applications.     

Relation between susceptibility to wheat bug attack and digestibility of glutenin

Two trials of winter wheat, embracing 17 varieties and 4 breeding lines, harvested in 2003 from locations in the semi-arid eastern part of Austria, were severely infested by naturally occurring bugs (Eurygaster sp.). In these trials bug damage was determined by selecting and weighing the infested kernels. Glutenin degradation was determined by the addition of meal from bug-infested kernels to meal from sound kernels from the same trials to reach sample mixtures representing a degree of bug attack of 6%. These mixtures and blanks made from sound meal were incubated in buffer solution (pH=8.5) at 37 °C for 45 min. The reduction in glutenin content due to the activity of bug proteinases was measured by RP-HPLC analysis of the glutenin fraction and comparison of the results of damaged and sound samples. The results showed good correlations between the degree of bug attack and the digestibility of glutenin (location 1: R²=0.69, P<0.01; location 2: R²=0.36, P<0.01). Both parameters seem to be variety specific. The determination of digestibility of glutenins was repeated in material from a trial harvested in 2005 in another location in eastern Austria, with eight varieties being the same as used in the 2003 trials. A correlation with the degree of bug attack in location 1 from the 2003 harvest was found again (R²=0.61, P<0.01).     

Milling and baking properties of field grown wheat expressing HMW subunit transgenes

Milling and baking tests were carried out on three transgenic wheat lines and their parental varieties grown in the field at two UK sites. The transgenic and control lines were essentially similar in their milling properties but the subunit 1Ax1 and 1Dx5 transgenes had different effects on breadmaking. The subunit 1Dx5 transgene resulted in a low loaf volume and poor crumb structure when expressed in lines with two or five endogenous HMW subunits, and this was accompanied by a greatly increased elastic modulus of the gel protein fraction. In contrast, the 1Ax1 transgene resulted in improved breadmaking quality and a more modest increase in the gel protein elastic modulus when expressed in the two subunit background. Blending of flour from a line expressing the 1Dx5 transgene with flour from a normal breadmaking wheat variety resulted in decreased breadmaking quality, even at a ratio of 1:9. The difference in the results obtained with the 1Ax1 and 1Dx5 transgenes may relate to the presence of an additional cysteine residue in the protein encoded by the latter, which promotes a more highly cross-linked glutenin network.     

Association analysis reveals effects of wheat glutenin alleles and rye translocations on dough-mixing properties

The glutenin loci of wheat (Triticum aestivum L.) are important determinants of bread-making quality, although the effects of alleles at those loci are incompletely understood. We applied an association analysis method to assess the effects of glutenin alleles and 1RS wheat–rye (Secale cereale L.) translocations on dough-mixing properties in 96 wheat cultivars and advanced lines grown at three Colorado locations while accounting for population structure and relatedness of individuals in the population. The results indicated that (1) in the majority of cases, controlling relatedness of individuals reduced the significance of associations between glutenin loci and Mixograph traits; (2) the Glu-D1 and Glu-B3 loci and 1RS translocations had greater impacts on dough-mixing properties compared to other glutenin loci; (3) Glu-B1w, Glu-D1d, and Glu-B3b were consistently associated with greater (more favorable) Mixograph peak time (MPT) than other alleles at the respective loci, whereas Glu-B1e, Glu-D1a, and Glu-B3c were associated with reduced MPT; (4) the 1BL.1RS translocation was associated with a decrease in Mixograph properties. Our results indicate that taking multiple-level relatedness of individuals into account can improve the results of association analysis for wheat-quality traits.     

Combined nitrogen and sulphur fertilisation and its effect on wheat quality and protein composition measured by SE-FPLC and proteomics

Soil sulphur deficiency, which is increasingly prevalent in Western Europe, lowers wheat yields, and also affects the gluten quality of the flour. Differences in S availability may change the proportion of S-poor to S-rich gliadins and glutenin subunits. This may cause unpredictable and unwanted variations in wheat quality. The combined effects of nitrogen (N) and sulphur (S) fertilisers and split application of S and N on wheat gluten quality and composition were investigated. The results revealed effects of S fertilisation on dough quality. At high N fertilisation levels significant responses to S fertilisation were found which emphasised the need for precision application of S in intensive wheat production systems. Protein fractionation by SE-FPLC showed that quality differences were associated with changing proportions of high M_r polymeric proteins. Changes in protein composition of salt soluble proteins were also confirmed by proteomics. Glyceraldehyde-3-phosphate dehydrogenase and one of the serpin protein spots increased at high N, combined with the lower S level. The enzymes also increased in samples with increased S fertilisation combined with low N, but was not changed at higher N levels. Furthermore, at high N the serpin protein spot, and also a 27 K protein and one unidentified protein spot decreased with increasing S.     

Physicochemical control of durum wheat grain filling and glutenin polymer assembly under different temperature regimes

Durum wheat is grown in the Mediterranean area where drought and high temperature frequently prevail and impact grain texture, composition and yield. The purpose of this work was to examine the effect of high temperature on grain development and final composition according to the timing of exposure. High temperature (up to 27.5 °C) was applied either during the linear grain filling or drying phases or during whole grain development. The dynamics of grain dry mass, water, glutenin polymers, and protein bodies during grain development were determined. Irrespective of high temperature timing, the arrest of grain filling was observed at 45.9% grain moisture content. At that point, starch granules included in endosperm cells reached their physical packing limit, limiting further deposits. HT applied before physiological maturity shortened the duration of grain filling and resulted in a significant increase in grain protein concentration and in the proportion of vitreous grain. Late formation of sodium dodecyl sulfate (SDS)-insoluble glutenin polymers below 32% grain moisture content was also favored. The ability of wheat storage protein to form a viscoelastic matrix embedding starch granules at the beginning of grain desiccation is proposed to be mandatory for gaining vitreous grains and a high proportion of SDS-insoluble glutenin polymers.     

Determination of wheat bug (Eurygaster spp.) damage in durum wheat (Triticum durum L.) by electrophoresis and rapid visco analyser

The degradation effects of wheat bug protease(s) on glutenin proteins of durum wheat cultivars were investigated by electrophoresis and modified rapid visco analyser (RVA) test. Glutenin patterns of the bug damaged durum wheats changed substantially due to bug protease(s). Although high molecular weight glutenin subunits (HMW-GS) of three cultivars (Ege, Svevo, and Zenith) disappeared after 60 min of incubation, the HMW-GS of other two cultivars (Diyarbakir and Firat) were still visible even after the longest incubation period at medium damage level. It shows that there was an intercultivar variation in susceptibility to hydrolysis by bug proteolytic enzymes. Low molecular weight glutenin subunits of all cultivars decreased substantially after 30 min of incubation. The RVA curves indicated a clear reduction in viscosity in semolina samples with both medium and high damage levels as compared to their respective undamaged (control) samples. There were significant correlations (p < 0.001) between bug damage level and viscosities at 3 min (r = −0.765), at 4.5 min (r = −0.549) and at 10 min (r = −0.835), breakdown value (r = −0.534) and decay rate (r = 0.600). Consequently, hydrolysis rate of wheat bug protease(s) can be determined by modified RVA technique without much more chemicals, procedures and expensive equipments.     

Composition of polymeric proteins and bread-making quality of wheat lines with allelic HMW-GS differing in number of cysteines

Three lines of Australian wheat variety Avocet, two biotypes of Australian variety Halberd, and the Italian bread wheat cultivar Fiorello and its derived line were used to study the possible role of the Glu-B1 HMW-GS in polymeric protein composition and flour functionality. These sets of lines contain HMW-GS 7+8, 7+9, 20x+20y or 26+27. Subunit Bx7 has four cysteine residues, while subunit Bx20 has only two. Compared to Avocet A (7+8), line Avocet C (20x+20y) exhibited an increase in polymeric protein, a decrease in the gliadin-to-glutenin ratio, and a marked decrease in unextractable polymeric protein (UPP). Avocet C doughs exhibited greater extensibility and shorter Mixograph dough development times, and baked into smaller loaves compared to those from Avocet A. Similar differences were observed in comparisons between the two Fiorello lines and between the two Halberd biotypes that had differing Glu-B1 alleles. These results show that in closely related genotypes, the presence of the Bx7+By8 or Bx7+By9 HMW-GS instead of Bx20+By20 or Bx26+By27 is associated with higher UPP, higher dough strength and better bread-making performance. It is suggested that this could be due to the greater number of cysteines in the Bx7 subunit that are available for forming higher-molecular-weight glutenin polymer. This could give the possibility to manipulate dough properties and bread-making quality by utilizing HMW-GS alleles with varying numbers of cysteine residues.     

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II' β-turns, and the latter a more compact globular structure rich in α-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol (TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.     

Isolation and characterisation of a xylanase inhibitor Xip-II gene from durum wheat

Cereals contain xylanase inhibitor proteins (XIPs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. Here, we report for the first time the isolation and characterisation of a genomic clone containing a xylanase inhibitor gene. This gene, Xip-II, isolated from a durum wheat genomic library (Triticum durum Desf.) encodes a mature protein of 307 amino acid (aa) residues that shares highest aa sequence identity (64%) with the rice RIXI xylanase inhibitor. XIP-II showed inhibition against family 11 xylanases and no chitinase activity. In silico analysis of the 5′ promoter region of Xip-II revealed sequences with similarity to known cis regulatory elements upstream from the initiation codon. In particular, the identification of a number of cis-acting elements controlling the expression of defence and seed-specific genes supports the role for this class of inhibitors in plant defence against pathogens but also provides new clues on a potential role in plant development.     

Molar fractions of high-molecular-weight glutenin subunits are stable when wheat is grown under various mineral nutrition and temperature regimens

Molar fractions of the high-molecular-weight glutenin subunits (HMW-GS) were determined for flour from bread wheat (Triticum aestivum L. cv Butte86) produced under 13 different combinations of temperature, water and mineral nutrition. Albumins, globulins and gliadins were removed from the flour by extraction with 0.3 M NaI in 7.5% 1-propanol. Total HMW-GS were recovered by extracting the remaining protein with 2% SDS and 25 mM DTT. Individual HMW-GS were then separated and quantified by RP-HPLC. Constant molar fractions for the five HMW-GS were maintained under all environmental conditions, despite large differences in duration of grain fill, total protein per grain, flour protein percentage, and total HMW-GS per grain. Similar molar fractions were found for five other US wheat varieties. The Bx7 subunit accumulated to the highest level at 30% of total HMW-GS. The Dx and Dy subunits were present in smaller but nearly equal proportions, 22% and 23%, respectively, and the Ax and By subunits were the least abundant, 14% and 12%, respectively. Although the amounts of HMW-GS per unit of flour are strongly affected by environment, the different subunits respond so similarly to external conditions that their final proportions appear to be determined mainly by genetic factors.     

Proteomic analysis of the mature kernel aleurone layer in common and durum wheat

The aleurone layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.     

影响小麦面团强度的贮藏蛋白基因表达研究

小麦品质主要由籽粒贮藏蛋白的含量和类型决定,高分子量麦谷蛋白亚基(HMW-GSs)组合是决定加工品质的主要因素。为了解HMW-GSs组合完全一致、蛋白含量相似的两个品种面团稳定时间相差近一倍的原因(郑麦158:高面团强度;郑麦369:低面团强度),通过转录组测序比较了籽粒发育中期3个时间点(花后14、21和28 d)的贮藏蛋白基因表达差异,分析了差异表达基因编码蛋白对面粉质量的贡献以及面粉的巯基含量差异等。结果表明,在3个时间点,两个品种间的HMW-GSs基因表达均无显著差异;郑麦158较郑麦369共有24个贮藏蛋白编码基因有显著表达差异,其中上调表达基因12个(23次),下调表达基因12个(23次)。12个显著上调表达基因中,包括9个燕麦类似蛋白基因(18次)、2个γ-醇溶蛋白基因(4次)和1个α-醇溶蛋白基因;在12个下调表达基因中,包括11个α-或α/β-醇溶蛋白基因(21次)和1个燕麦类似蛋白基因(2次)。两品种比较,郑麦158面粉的硫元素含量较低,但自由巯基和二硫键含量较高。基于蛋白二硫键预测和面粉蛋白质量评价模型分析结果,差异表达基因编码的燕麦类似蛋白和γ-醇溶蛋白对面团强...     

Influence of gliadin removal on strain hardening of hydrated wheat gluten during equibiaxial extensional deformation

The aim of the present work has been to study the equibiaxial extensional deformation of doughs of gluten- and glutenin-rich fractions containing 40 wt% water subjected to lubricated squeezing flow with four different crosshead speeds at room temperature. The gluten dough shows strain softening and hardening in succession whilst the dough where the gliadins have been removed by alcohol extraction does not show strain hardening behavior but breaks immediately after strain softening. The equibiaxial extensional viscosity decreases with increasing strain rate at given strains, appearing as strain rate thinning behavior, which is stronger in the glutenin dough than in the gluten dough. The large extensibility with strain hardening in the gluten dough is due to the presence of gliadins acting as both plasticizers and promoters for the more extensible networks.     

小麦谷蛋白亚基及其基因多态性研究进展

本文主要介绍了小麦高、低分子量谷蛋白亚基及其控制基因的研究进展情况,包括各种不同的研究技术以及相应的研究结果。除了传统的SDS-PAGE技术以外,分子标记技术以及免疫化学技术都在该领域中得到日益广泛的应用,并取得了关于亚基及基因的多态性和快速鉴别方法等一系列重要的研究进展。     

A micro-method for the determination of Yellow Pigment Content in durum wheat

Colour is an important component of durum wheat quality affecting consumer’s choice. Pasta colour is the result of the carotenoid pigments accumulated in the seed endosperm and of the enzymatic activities responsible for their degradation. Thus, increasing yellow colour is a valuable goal for improving the commercial and nutritional values of durum wheat products. This work reports the set up of a micro-method for the determination of Yellow Pigment Content (YPC) in durum wheat. The novel method, although similar in principle to the Official Method AACC 14-50, allows the use of micro-amount of samples (10–100 mg), a condition that for durum wheat corresponds to a single kernel, and micro-amount of extraction solvent (water-saturated 1-butanol, 250–500 μL). These improvements together with a short extraction time lead to significant benefits from the practical and the economic point of view. The improved method has been validated using nine durum wheat cultivars extracted in 15 min with the ultrasonic bath. The YPC was calculated by reading the absorbance at 435 nm after the manual injection of an aliquot of extract directly into a UV–Vis HPLC detector. The results obtained were comparable and not significantly different from the Official AACC Method 14-50.     

Chemical characterization and biological effects of immature durum wheat in rats

A durum wheat cultivar, Simeto, was grown in experimental fields and samples were collected at various stages of development (from 9 to 45 days after flowering) to assess changes in chemical composition during maturation. Fructans, in particular low molecular weight fructo-oligosaccharides, accumulated in the first 2–3 weeks after anthesis. The cultivar was then grown in an open field and collected at 15 days after anthesis and at maturity 45 days after anthesis. Experimental diets containing 53% wholemeal from immature or mature wheat, were fed for 6 and 12 weeks to two groups of growing rats. Glutathione, vitamins C and E and total hydrophilic or lipophilic antioxidant concentrations were determined in mature and immature wholemeal. The effects of feeding immature and mature wheat diets for the two experimental periods on the immune system, antioxidant status and plasma lipids were studied. Feeding immature wheat increased the proliferation rates of lymphocytes, indicating a stimulating effect on the immune response, decreased the plasma triglycerides and cholesterol levels, indicating a positive effect on lipid profiles. Antioxidant concentrations in blood and lymphocytes did not change significantly.     

Characterisation of a specific class of typical low molecular weight glutenin subunits of durum wheat by a proteomic approach

Glutenin polymers are formed by high (HMW-GS) and low molecular weight glutenin subunits (LMW-GS). The latter group of subunits has been less characterised compared to the former due to their great number and heterogeneity.