Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.     

Modification of the Low Molecular Weight (LMW) Glutenin Composition of Transgenic Durum Wheat: Effects on Glutenin Polymer Size and Gluten Functionality

The low-molecular weight (LMW) glutenin subunits are major determinants of the viscoelasticity of durum wheat gluten, and therefore of its technological quality, with both quantitative effects and qualitative effects. We have modified the LMW glutenin subunit composition of the durum wheat cultivar Ofanto by expression of a transgene encoding a B-type LMW glutenin subunit and have carried out detailed analyses of two independent transformed lines in order to assess the effect of the transgene on the size distribution of the glutenin polymers and on their functional properties. In one line the expression of the transgene led to an increase in the amount of large glutenin polymers resulting in stronger and more stable dough. In the second line, however, the expression of the transgenic subunit was accompanied by decreased expression of endogenous LMW subunits with consequent detrimental effects on glutenin polymers and dough viscoelasticity. These results demonstrate that the LMW glutenin subunits contribute to the functional properties of wheat by influencing the amount and the distribution of glutenin polymers and indicate that either plant breeding or GM technology can be used to 'fine tune' the properties of durum wheat for different end uses by manipulating the amount and structures of individual LMW subunit proteins.     

小麦高分子量麦谷蛋白亚基的遗传变异与小麦加工品质改良

高分子量麦谷蛋白亚基（HMW-GS）是小麦胚乳中一种具有多态性的蛋白质组分,在面团中它们可以通过相互之间或与低分子量麦谷蛋白亚基（LMw-Gs）之间形成二硫键来组成麦谷蛋白多聚体。由于其在小麦面粉加工所需的粘性和弹力方面具有极其重要的作用,过去几十年间在小麦加工品质相关蛋白研究方面的工作大多数集中在高分子量麦谷蛋白亚基上。近几年在高分子量麦谷蛋白亚基及其编码基因的鉴定、基因的遗传变异以及不同变异在小麦加工品质中的作用方面进行了大量研究。本文对近几年在HMW-GS领域的研究进展进行综述并且重点讨论HMW-GS的变异及其对小麦品质育种的重要意义。     

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.     

基因枪法转化小麦谷蛋白基因研究进展

小麦面粉品质的优劣主要取决于麦谷蛋白多聚体结构的组成,谷蛋白多聚体由高分子量谷蛋白亚基(HMW-GS)、低分子量谷蛋白亚基(LMW-GS)和醇溶蛋白以二硫键相互交联构成,其数量和结构特征直接影响面团的粘弹性,所以通过基因工程方法转化优质谷蛋白基因,增加谷蛋白数量,改善谷蛋白多聚体结构组成,进而改良面粉品质的研究逐渐引起国内外的重视,并在近年来取得了重要进展。基因枪法是目前利用基因工程改良小麦品质的主要途径,自1992年以来已在多个研究室取得了较为瞩目的成果,显示了基因工程改良小麦品质的可能性及前景。综述了迄今为止国内外利用基因枪法转化谷蛋白基因改良小麦品质的研究进展,并在受体材料的选择等方面的研究现状作了较为详细的阐述。     

Analysis of dough functionality of flours from transgenic wheat

The rheological properties of flours from five different lines of transgenic wheat that either express or over-express subunits 1Ax1 and 1Dx5 were analyzed by mixograph assays and SDS sedimentation tests. In one case, the over-expression of subunit 1Dx5 resulted in a ca. 2-fold increase in mixing time, associated with a significant improvement in dough strength, and a lower resistance breakdown, suggesting an important increase in dough stability. However, the flour failed to develop properly without mixing with control flour because the rate of mixing was insufficient to develop the dough, i.e., the flour was overstrong. In two wheat transgenic lines, the expression of 1Ax1 and 1Dx5 transgenes, associated with silencing of all the endogenous high-molecular-weight glutenin subunits, resulted in flours with lower mixing time, peak resistance and sedimentation volumes, suggesting a lower gluten strength.     

Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves flour mixing properties

Avenin-like b proteins may contribute to the viscoelastic properties of wheat dough via inter-chain disulphide bonds, due to their rich cysteine residues. In order to clarify the effect of the avenin-like b proteins on the functional properties of wheat flour, the functional and biochemical properties of wheat flour were analyzed in three transgenic wheat lines overexpressing the avenin-like b gene using the sodium dodecyl sulfate sedimentation (SDSS) test, Mixograph and size exclusion-high performance liquid chromatography (SE-HPLC) analysis. The results of the SDSS test and Mixograph analysis demonstrated that the overexpression of avenin-like b proteins in transgenic lines led to significantly increased SDSS volume and improved flour mixing properties. The results of SE-HPLC analysis of the gluten proteins in wheat flour demonstrated that the improvement in transgenic line flour properties was associated with the increased proportion of large polymeric proteins due to the incorporation of overexpressed avenin-like b proteins into the glutenin polymers. These results could help to understand the influence and mechanism of avenin-like b proteins on the functional properties of wheat flour.     

Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene

It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties. In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber. The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer. Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers. Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber. To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots. The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.     

Coexpression of the High Molecular Weight Glutenin Subunit 1Ax1 and Puroindoline Improves Dough Mixing Properties in Durum Wheat (Triticum turgidum L. ssp. durum)

Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS), plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina) and Puroindoline b (Pinb) genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread) and may improve the economic values of durum wheat.     

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.     

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.     

小麦低分子量麦谷蛋白亚基研究进展

低分子量麦谷蛋白亚基(LMW-GS)是小麦胚乳中的一种聚合蛋白组分,LMW-GS彼此间或／和高分子量麦谷蛋白亚基(HMW-GS)间形成分子内二硫键,进而产生麦谷蛋白聚合体,决定小麦面团的加工品质。由于 LMW-GS与醇溶蛋白的提取特性和电泳迁移率相近,其研究进展缓慢。近年来随着电泳技术的提高,LMW-GS的研究也成为品质性状研究的新热点,越来越多的研究证实了LMW-GS对品质具有重要作用。然而,关于LMW-GS 的研究在我国尚处于起步阶段。本文从小麦LMW-GS的分类、染色体定位、结构及其与品质间关系等方面回顾其研究状况,并讨论研究中存在的问题。     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

Overexpression of Avenin-Like b Proteins in Bread Wheat (Triticum aestivum L.) Improves Dough Mixing Properties by Their Incorporation into Glutenin Polymers

Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.     

小麦高分子量麦谷蛋白亚基序列及其结构与加工品质的关系

高分子量麦谷蛋白亚基（high molecular weight glutenin subunit,HMW-GS）是小麦种子贮藏蛋白的主要成分,其组成、含量和结构直接影响小麦面粉面筋的弹展性,决定着小麦的加工品质。本文主要对小麦HMW-GS的序列、结构和亚基之间组合形式做了详细的综述,并较系统地讨论了HMW-GS的结构和组成、特点等与面粉的加工品质之间的关系以及如何从定性和定量两方面来影响面粉的加工品质。     

花后灌水次数对强筋小麦籽粒产量和品质的影响

在防雨池栽培条件下,以2个优质强筋小麦品种（济麦20和藁城8901）为试验材料,研究了花后不同水分供应状况对籽粒产量、籽粒品质（粉质仪参数和面包体积）及其蛋白质组分的影响.结果表明：2个品种的籽粒产量、面粉的面团形成时间、面团稳定时间和制成面包体积均随花后灌水次数的增加呈先升高后降低的趋势;其中,花后灌1水（花后7 d）时藁城8901的籽粒产量最高,花后灌2水（花后7 d+花后14 d）时济麦20的籽粒产量最高;2个品种面粉的面团形成时间、面团稳定时间和制成面包体积均以花后灌1水时最优.单体蛋白含量、不溶性谷蛋白含量、谷蛋白总含量、蛋白质含量以及湿面筋含量也呈现类似的变化趋势.逐步回归分析表明,花后不同水分供应状况下,不溶性谷蛋白含量是影响面团稳定时间的关键因素,谷蛋白总含量与面包体积的变化密切相关.因此,为了保持优质强筋小麦品质的稳定性,水分管理应以改善籽粒蛋白质特别是谷蛋白组分的构成为目标.     

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.Communicated by J. W. Snape     

The structure and properties of gluten: an elastic protein from wheat grain

The wheat gluten proteins correspond to the major storage proteins that are deposited in the starchy endosperm cells of the developing grain. These form a continuous proteinaceous matrix in the cells of the mature dry grain and are brought together to form a continuous viscoelastic network when flour is mixed with water to form dough. These viscoelastic properties underpin the utilization of wheat to give bread and other processed foods. One group of gluten proteins, the HMM subunits of glutenin, is particularly important in conferring high levels of elasticity (i.e. dough strength). These proteins are present in HMM polymers that are stabilized by disulphide bonds and are considered to form the 'elastic backbone' of gluten. However, the glutamine-rich repetitive sequences that comprise the central parts of the HMM subunits also form extensive arrays of interchain hydrogen bonds that may contribute to the elastic properties via a 'loop and train' mechanism. Genetic engineering can be used to manipulate the amount and composition of the HMM subunits, leading to either increased dough strength or to more drastic changes in gluten structure and properties.     

The low-molecular-weight glutenin subunit proteins of primitive wheats. IV. Functional properties of products from individual genes

 Three genes encoding the low-molecular-weight glutenin subunits (LMW-GSs), LMWG-E2 and LMWG-E4, from A-genome diploid wheat species, and LMW-16/10 from a D-genome diploid wheat, were expressed in bacteria. The respective proteins were produced on a relatively large scale and compared with respect to their effects on flour-processing properties such as dough mixing, extensibility and maximum resistance; these are important features in the end-use of wheat for producing food products. The LMWG-E2 and LMWG-E4 proteins caused significant increases in peak resistance and mixing time, compared to the control, when incorporated into dough preparations. The LMWG-16/10 protein was qualitatively less effective in producing these changes. All three proteins also conferred varying degrees of decrease in dough breakdown. LMWG-E2 and LMWG-E4 caused significant increases in dough extensibility, and decreases in maximum resistance, relative to the control. LMW-16/10 did not show a significant effect on extensibility but showed a significant decrease in maximum resistance. The refinement of relating specific features of the structure of the LMW-GS genes to the functional properties of their respective proteins is discussed. Received: 24 November 1997 / Accepted: 18 August 1998     

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer

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.