Perhaps the first successful research directed at improving protein quality in cereals was that of increasing the lysine content in maize (Glover and Mertz, 1987; Mertz et al., 1964). The discovery that the opaque2 (o2) mutation increased the lysine content of maize endosperm by decreasing the synthesis of prolamin (zein) proteins and increasing the level of other types of endosperm proteins prompted a search for similar mutants in other cereal species (Munck, 1992). Unfortunately, the low seed density and soft texture of this type of mutant were associated with a number of inferior agronomic properties, including brittleness and insect susceptibility. With only a few exceptions (Habben and Larkins, 1995), these mutants were not commercially developed. However, the subsequent identification of genetic modifiers (suppressors) that create a normal kernel phenotype while maintaining the higher lysine content caused by the o2 mutation in maize permitted the development of a new type of o2 mutant known as quality protein maize (QPM) (Prasanna et al., 2001). QPM is currently being grown in several developing countries, where it is helping to alleviate protein deficiencies.
Other approaches to increase the lysine content of maize seed include site-directed mutagenesis of genes encoding the major prolamin proteins, α- and γ-zeins. As previously described, zeins are asymmetrically organized in ERlocalized PBs, such that the most hydrophobic proteins, α-zeins, are found in the center and the more hydrophilic γ-zeins are at the periphery (Lending and Larkins, 1989). As zeins are essentially devoid of lysine (Woo et al., 2001), the question arises as to whether the addition of such charged amino acids will disrupt the way in which zeins form accretions within the ER. Wallace et al. (1988) demonstrated the consequence of inserting lysine residues into different regions of a 19-kDa α-zein protein. When the modified proteins were synthesized in Xenopus oocytes, they formed accretions similar to the native proteins, suggesting that the presence of lysine was not detrimental to their aggregation and deposition. It was shown that green fluorescent protein insertions into a 22-kDa a-zein protein did not disrupt PB formation in yeast cells (Kim et al., 2002). This observation suggests that α-zeins can be subjected to substantial structural modification and still aggregate into insoluble accretions.
A similar approach was taken with the sulfur-rich 27-kDa γ-zein. It was first demonstrated that 27-kDa γ-zein accumulates in ER-derived PBs in Xenopus oocytes and Arabidopsis (Geli et al., 1994; Torrent et al., 1994). When various modified versions of the protein were expressed in Arabidopsis, it was found that the N-terminal domain is necessary for ER retention and the C-terminal domain is necessary for PB formation. However, the central domain could be replaced with lysine-rich polypeptides without affecting protein stability and targeting (Geli et al., 1994). These lysine-rich γ-zeins were also shown to accumulate to high levels in association with endogenous α- and γ-zeins in transiently transformed maize endosperm cells (Torrent et al., 1997). Thus, the addition of lysine and other charged amino acids to α- and γ-zein proteins does not appear to alter their structural properties sufficiently to inhibit assembly into PBs. However, the consequences of these changes when the genes are expressed in stably transformed corn plants remain to be described. Another important question is whether sufficient levels of these proteins can be accumulated to make a significant increase in endosperm lysine content.
Rice contains very little prolamin; its major storage protein, a so-called glutelin, is a highly insoluble 11S globulin (Table 5.1). This protein is lysine deficient, whereas 11S globulins in legumes are deficient in sulfur-containing amino acids. Consuming both rice and legumes can provide an adequate balance of these essential amino acids, and this is especially important in vegetarian or low meat diets. Consequently, the expression of legume globulins in rice is one strategy for improving its amino acid balance. The gene encoding proglycinin, the precursor of soybean 11S globulin, was modified by replacing a variable region of amino acid sequence with a peptide encoding four contiguous methionine residues (Kim et al., 1990). The genetically engineered protein was found to be stably accumulated in Escherichia coli cells (Kim et al., 1990). In plant tissues, the modified glycinin accumulated to a similar degree as the mature protein and in the correct conformation (Utsumi et al., 1993, 1994). For example, using the class 1 patatin promoter, tuber-specific expression of the modified glycinin, amounting to 0.2–1% of total protein, was achieved in transgenic potato (Utsumi et al., 1994). The methionine-enriched and unmodified glycinins were transformed into rice under control of the promoter of the glutelin, GluB-1, which is one of the most highly expressed genes in rice endosperm (Katsube et al., 1999). In transgenic rice, assembly of proglycinin into 7–8S trimeric structures, cleavage into acidic and basic subunits, and assembly into 11–12S hexameric structures in storage vacuoles all occurred in a manner similar to that in soybean. The endogenous glutelins formed 11S complexes with glycinins, indicating the transgenic protein did not adversely affect the assembly or accumulation of native storage proteins (Katsube et al., 1999). Soybean glycinins have the property of lowering human serum cholesterol levels, and this fact offers an advantage for expression in rice, in addition to it being able to increase the lysine and, potentially, methionine contents (Kito et al., 1993). Pea legumin, which is higher in lysine than rice glutelin, has also been expressed in rice endosperm in an effort to improve its amino acid composition (Sindhu et al., 1997).
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