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  Section: Molecular Biology of Plant Pathways » Genetic Engineering of Seed Storage Proteins
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Modification of Grain Biophysical Properties

In the developed world, optimization of seed protein quality is more important for livestock feed than for human diets. Indeed, the vast majority of world grain consumption is in livestock rations. With the exception of rice, human grain consumption is mainly through processed foods, and optimization of particular processing characteristics for specific end uses is of paramount importance. Wheat, in particular, is mainly used as white flour, which after removal of the germ and the bran is essentially composed of starch and gluten proteins. The amount and composition of the gluten determines end use, with high-gluten flours primarily being used for bread and pasta making (Shewry et al., 2003a). The storage proteins comprising the gluten form insoluble accretions in endosperm cells of the wheat grain, but when mixed with water they create viscoelastic matrices that are essential in the bread leavening process. The HMW-glutenin subunits (HMW-GSs) are considered to be the most important components of gluten and have been subjected to structural modification for studying their function and bread-making characteristics (Shewry and Halford, 2002). For a comprehensive review of the role of glutenins in determining wheat processing properties, the reader is directed to a review by Shewry et al. (2003a).

Large-scale bacterial expression allowed the production of homogeneous HMW-GSs, which is necessary for detailed structure––function analyses (Dowd and Bekes, 2002; Galili, 1989). Other studies expressing modified glutenins were directed at systematically dissecting the functional domains of these proteins (Anderson et al., 1996; Shimoni et al., 1997).

Research aimed at upregulating HMW-GSs in wheat developed in part from the demonstration that differences in gluten properties are due to allelic variation in the composition of HMW-GS (Payne, 1987). Cultivars of hexaploid bread wheat have six genes encodingHMW-GSs, with differences in gene expression resulting in variable amounts of these proteins (Shewry and Halford, 2002). Ectopic expression of genes encoding the 1Ax and 1Dx5 subunits led to variable accumulation of the transgenic proteins and, where studied, variable effects on gluten strength (Altpeter et al., 1996; Alvarez et al., 2000; Barro et al., 1997; Blechl and Anderson, 1996; Popineau et al., 2001). Several transgenic lines exhibiting stable expression of 1Ax1 driven by its own promoter have been characterized in detail following field trials
(Vasil et al., 2001). There was no evidence that expression of an extra HMW-GS gene resulted in gene silencing or any undesirable effect on yield, protein composition, or flour functionality, and in some of the transgenic lines, mixing time, loaf volume, and water absorbance improved relative to the control cultivar (Vasil et al., 2001). However, in at least one other study, gene silencing of endogenous subunits was encountered (Alvarez et al., 2000). The expression of 1Ax1 and 1Dx5 transgenes caused silencing of all the endogenous HMW-GSs, and rheological analysis showed a lower dough strength (Alvarez et al., 2001). In the nonsilenced lines, a direct correlation was found between the number of HMW-GS genes expressed and bread dough elasticity (Barro et al., 1997). One line overexpressing the 1Dx5 subunit exhibited a significant improvement in dough strength. In fact, it was necessary to mix the flour with a low gluten, soft flour in order to allow adequate mixing and dough development (Alvarez et al., 2001). Similarly, very strong glutens giving rise to doughs with unusual mixing characteristics were obtained with a transgenic line overexpressing 1Dx5, in comparison to a nearly isogenic line expressing 1Ax1 that had little effect (Popineau et al., 2001). While both lines accumulated the transgenicHMW-GS protein at 50–70% of total HMW glutenin and exhibited
increased glutenin aggregation, only the 1Dx5 transgenic line exhibited increased dough elasticity resulting from increased glutenin cross-linking (Popineau et al., 2001). The possibility of using the viscoelastic properties of glutenins to produce novel dough characteristic in maize is being investigated (Sangtong et al., 2002). The 1Dx5 HMW-GS was shown to be stably expressed and genetically transmitted in maize (Sangtong et al., 2002), and experiments to test the viscoelastic properties of doughs produced from such transgenic lines are under way.

There is substantial evidence to suggest that disulphide cross-linking is important in stabilizing the wheat glutenin backbone (Shewry and Tatham, 1997). Presence of the 1Bx20 HMW-GS in pasta wheat (Triticum durum) is associated with poor pasta-making quality (Liu et al., 1996), and when present in bread wheat, it is associated with poor bread-making quality (Payne, 1987). This subunit has been sequenced and compared to the highly similar 1Bx7 HMW-GS (Shewry et al., 2003b). 1Bx7 confers increased dough strength compared with 1Bx20 and contains two N-terminal cysteines, which are substituted with tyrosine residues in 1Bx20. Therefore, the poor dough-making properties conferred by 1Bx20 are thought to be due to its reduced ability to cross-link with the gluten network (Shewry et al., 2003b). This may be the reason to target this HMW-GS for transgenic downregulation. Many studies have demonstrated the feasibility of manipulating the properties of individual glutenin subunits in order to affect gluten structure but much remains to be learned about the interactions involved.

Although theHMW-GSs form the backbone of the elastomeric gluten network, the interaction of other glutenins and gliadins is believed to be important. A new family of low-molecular weight gliadins was reported (Clarke et al., 2003). Sequence analysis and genetic mapping revealed homology to a 17-kDa barley protein involved in beer foam stability and a different chromosomal location in wheat from that of the glutenins and gliadins. Purification of an E. coli-expressed member of this family and incorporation into a base flour produced a stronger dough with a substantial increase in bread loaf height (Clarke et al., 2003). This demonstrates the importance of other types of wheat storage proteins in gluten formation and suggests that such proteins may be suitable for transgenic modification to improve bread-making characteristics.

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