Interest in improving the nutritional quality of cereals was stimulated by the discovery in the 1960s and 1970s of a range of high-lysine mutants in maize, sorghum, and barley. The first of these were the opaque2 (21) and floury2 (22) mutants of maize, followed by the lys gene of Hiproly barley (23) and hi mutant of sorghum (24). These are all spontaneously occurring mutant genes; a range of other spontaneous high-lysine genes were subsequently identified in maize (17), and a range of chemical and physical mutagens were used to generate further high-lysine genes in sorghum (25) and barley (26,27). Despite initial optimism, only one of these mutant genes, opaque2 of maize, has been successfully exploited in plant breeding programs, using genetic modifiers to convert the soft starchy endosperm to a normal type (17). Although the other mutants have failed to be exploited because of associated deleterious effects on yield, analysis of their composition and molecular basis has contributed to our understanding of the regulation of seed protein biosynthesis and allowed us to make more informed decisions on strategies for improving quality.
The high-lysine phenotype of most mutant lines is due, at least in part, to a reduction in the proportion of the lysine-poor prolamins and increases in other more lysine-rich proteins. It is, therefore, tempting to suggest that high-lysine cereals could be engineered by down-regulation of prolamin gene expression using antisense or cosuppression technology, as described for 2S albumins (napins) and globulins in transgenic oilseed rape (28,29). In this work, antisense technology was used to down-regulate either napin or 12S globulin gene expression in developing seeds, resulting in compensatory increases in the remaining protein group. However, in all high-lysine cereals the decreased prolamin content is associated with reduced starch synthesis and hence lower yield, and this has proved difficult to separate from the high-lysine character by plant breeding. Consequently, reduction in prolamins is not generally considered to be a valid strategy for engineering high-lysine cereals. Instead, three different strategies have been proposed.
A. Manipulation of the Amino Acid Composition of Prolamins
Wallace et al. (30) designed modified maize prolamins (zeins) containing single and double substitutions of lysine for neutral amino acids; insertions of oligopeptides (five to eight residues) rich in lysine, threonine, and tryptophan; and insertion of an Mr 17,000 peptide derived from the SV40 VP2 protein. All except for the latter (included as a control) were synthesized and aggregated into dense particles when the messenger RNA (mRNA) was injected into egg cells of the frog Xenopus, indicating that the proteins should also be stable in transgenic cereals. However, the drawback of this strategy is that zeins account for about half of the total protein in the maize seed and appear to be encoded by up to 100 genes (17). Consequently, it would be necessary to express the mutant zeins under control of a highly active promoter and possibly also to combine this with down-regulation of the endogenous wild-type zeins. A similar strategy could be applied to other cereals.
Detailed analyses of the high-lysine barley mutant Hiproly demonstrated that about half of the increase in lysine resulted from elevated levels of four specific lysine-rich proteins, which together account for about 17% of the total grain lysine, compared with about 7% in normal lines (31). These proteins are /3-amylase (=5.0 g % Lys), protein Z (now known to be a serpin proteinase inhibitor) (—7.1 g % Lys), and chymotrypsin inhibitors CI-1 (—9.5 g % Lys) and CI-2 (=11.5 g % Lys) (31).
CI-1 and CI-2 were both purified from Hiproly, providing a basis for molecular cloning. This demonstrated that both proteins occur in two iso-forms (32) with the major form of CI-2 comprising 84 residues with an Mr of 9,380. This form contains seven lysine residues (8.3 mol %) but no cysteine residues (and hence no disulfide bonds) (33). The three-dimensional structure of the protein shows a wedge-shaped disc of about 28 X 27 X 19 A with a single «-helix of 3.6 turns and four strands of )S-sheet with a left-handed twist (Fig. 1). The reactive (inhibitory) site (Met-59) of CI-2 is present on an exposed nine-residue loop (Gly-54 to Arg-62). Perutz and coworkers (34,35) have shown that it is possible to mutate or extend this loop region, inserting up to 10 glutamine residues at Met-59 in order to explore the role of glutamine repeats in neurodegenerative diseases. More recently,
Roesler and Rao (36) have described the design and expression in E. coli of five mutant forms of CI-2 containing 20-25 mol % lysine, based mainly on mutation of surface-exposed residues. Although all the mutants appeared to have conformations similar to that of the wild-type protein, as determined by circular dichroism spectroscopy, they had lower thermodynamic stabilities. Nevertheless, at least one of the mutants was considered to be suitable for expression in transgenic plants.
The thionins are another group of lysine-rich seed proteins (37). Rao et al. (38) designed high-lysine analogues of barley a-hordothionine, a 45-residue protein containing five lysines (11 mol %). These included an analogue containing seven additional lysines (i.e., a total of 27 mol %), which was synthesized and shown to exhibit antifungal activity similar to that of the wild-type protein. The authors have not, however, attempted to express any of the mutant proteins in transgenic plants, and such material would perhaps be unlikely to be accepted by consumers or regulatory authorities because of the wide biological activity of thionins (37).
Pea legumin contains about 5 mol % lysine (39), which is similar to the nutritional requirements for humans (5.5 g %) (Table 1). Sindhu et al. (40) reported that pea legumin was stably accumulated in the seeds of transgenic rice plants but did not determine the effect on the lysine content of the grain.
An alternative approach to using naturally occurring high-lysine proteins, or mutant forms of these, is to design high-quality proteins ab initio. Keeler et al. (41) demonstrated the feasibility of this concept by designing low-Mr proteins (3600 to 6700) with an a-helical coiled coil structure and containing up to 43 mol % lysine accompanied by high levels of methionine and in some cases also tryptophan. The proteins were initially synthesized in E. coli and one protein containing 31 mol % lysine and 20 mol % methionine was then expressed in tobacco seeds under control of the bean phaseolin and soybean /3-conglycinin promoters. Increases in lysine of up to 0.8 mol %, compared with a wild-type level of 2.56 mol %, were observed in the primary transformants and were inherited through three generations. No impact on seed methionine levels was reported, and the work has not so far been extended to cereals or other crop plants.
The most spectacular success in increasing the lysine content of cereals has been achieved by increasing the amount of free rather than protein-bound lysine. This is based on eliminating the feedback regulation that normally limits the pool of free lysine by transforming with genes for feedback-insensitive enzymes from microorganisms. Two enzymes are important in this
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