Improving the protein content and composition of cereal grain

ARTICLE IN PRESS Journal of Cereal Science 46 (2007) 239–250 www.elsevier.com/locate/jcs Improving the protein content and composition of cereal grain Peter R Shewryà Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Received 17 October 2006; received in revised form 23 March 2007; accepted 10 June 2007 Abstract Cereals are important sources of protein for human nutrition but have low quality due to limitations in the amounts of essential amino acids, notably lysine These deficiencies result from the low levels of these amino acids in the prolamin storage proteins and hence are exacerbated when high levels of nitrogen fertiliser are used to increase yield and total protein content Genetic and genetic engineering strategies to increase both total protein content and the composition of essential amino acids have been employed These include the exploitation of mutant high lysine genes and the use of transformation to either express additional proteins which are rich in lysine and/or methionine or to increase the free pools of these amino acids r 2007 Elsevier Ltd All rights reserved Keywords: Cereals; Lysine; Methionine; Protein content; Essential amino acids; Nutritional quality Introduction Cereals are a major source of dietary protein for humans FAO production figures for the years 2001–2005 (http://faostat.fao.org) show that the mean annual production of all cereals exceeded 2100 million tonnes, with the three major species (maize, wheat, rice) accounting for about 85% of this Even at a conservative estimate of 10% protein, this equates to over 200 million tonnes of cereal grain protein harvested each year, with a substantial proportion of this being consumed by humans The diets consumed in developed countries usually contain various sources of dietary protein (cereals, legume seeds, meat, etc.) and the compositions of individual dietary components are of little real importance in relation to nutritional requirements However, this is not the case in some less developed countries in which a single cereal may Abbreviations: AK, aspartate kinase; CI-1, chymotrypsin inhibitor-1; CI-2, chymotrypsin inhibitor-2; DHDPS, dihydrodipicolinate synthase; EAA, essential amino acid; GM, genetic manipulation; GPC, grain protein content; HDH, homoserine dehydrogenase; LKR, lysine ketoglutarate reductase; ND, not determined; QPM, quality protein maize; QTLs, quantitative trait loci; RFLP, restriction fragment length polymorphism; SDH, saccharopine dehydrogenase ÃTel.: +44 1582 763133; fax:+44 1582 763010 E-mail address: peter.shewry@bbsrc.ac.uk 0733-5210/$ - see front matter r 2007 Elsevier Ltd All rights reserved doi:10.1016/j.jcs.2007.06.006 account for a major part of the total protein intake In this case, the nutritional quality (i.e content of essential amino acids) of the protein as well as the amount may be important Similarly, the protein composition is important when formulating feed for livestock in developed countries, particularly fast growing monogastric animals such as pigs and poultry Improving the quality for these animals has therefore been the major driver for much of the work on cereal protein quality in developed countries Grain protein content (GPC) Because of their different production systems and levels of fertiliser use, it is difficult to obtain comparative values for the protein contents of different cereals However, consideration of values reported indicates that relatively small differences exist within and between species and that these are amplified by environmental factors Thus, ranges of 5.8–7.7% of protein on a dry weight basis have been quoted for rice (Champagne et al., 2004); 8–15% for barley (Shewry, 1993) and 9–11% for maize (Zuber and Darrah, 1987) However, these ranges almost certainly reflect the impacts of environment as well as genotype For example, Kirkman et al (1982) showed that grain nitrogen varied from 1.27% to 2.01% (about 7.2–11.5% protein) in a single variety of barley in which N fertilisation and cropping ARTICLE IN PRESS 240 P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 history were varied and there are numerous similar reports in the literature Nevertheless, genetic control of GPC is well established, with a classic example being the long-term Illinois selection experiment in which 70 generations of selection resulted in lines of maize with protein contents ranging from 4.4% to 26.6% (Dudley et al., 1974) There is limited interest in high protein maize as much of the crop is used for livestock feed as a mixture with soybean and other protein sources However, more research has been carried out in two species in which protein content underpins grain quality: barley and wheat Although some barley is consumed by humans the vast majority is either used for livestock feed or for malting, brewing and distilling These uses have different requirements, with low protein being required for malting and higher protein for feed, leading to the breeding of cultivars which vary in their protein content when grown under the same agronomic conditions Ullrich (2002) has provided an excellent review of the genetic control of GPC in barley The trait is clearly polygenic in this species with quantitative trait loci (QTLs) being mapped onto all seven chromosomes These loci vary in their contributions to the variation in protein content in individual crosses but Ullrich (2002) concluded that QTLs were particularly concentrated on chromosomes (2H), (4H) and (5H) 2.1 Grain protein content (GPC) in wheat Whereas the major emphasis in barley has been on low protein for malting, the emphasis in wheat has been on high protein wheats for nutritional enhancement and improved processing performance Johnson et al (1985) have provided an excellent overview of earlier studies of sources of high protein in wheat Initial screening of the USDA World Wheat Collection showed that the protein content of 12,600 lines varied from about 7% to 22% (Vogel et al., 1978), with the genetic component accounting for about a third of this (i.e about 5%) Hence, the greater part of the variation was due to non-genetic factors and this strong environmental impact has made breeding for high protein difficult Nevertheless, genetic sources of high protein have been identified with Atlas 50 and Atlas 66 being derived from the South American cultivar Frondoso (Johnson et al., 1985) and Nap Hal from India (www.indiaresource.org) These sources appear to have different ‘‘high protein genes’’ and crosses between Atlas 66 and Nap Hal showed transgressive segregation for protein content Both lines were used extensively for crosses in the Nebraska wheat breeding programme and the Atlas 66 genes were successfully transferred into the hard red winter wheat Lancota which had good quality and agronomic performance Frondoso and related Brazilian lines which probably have the same high protein gene(s) were also incorporated into hard red spring wheats suitable for the northern part of the USA (Minnesota, Montana, North and South Dakota) (Busch and Rauch, 2001) Although high protein has not been a major target in the UK, selection for breadmaking performance has resulted in a difference between the protein contents of UK breadmaking and feed wheats, illustrated by Avalon and Hobbit, respectively, which differ in their protein contents by about 2% (Snape et al., 1993) Wider sources of genes for high protein have also been identified and exploited For example, the Kansan variety Plainsman V contains a gene(s) from Aegilops which is thought to increase GPC by 2–3% (Finney, 1978) However, the most widely exploited sources of high protein genes in wheat are wild emmer (Triticum turgidum var dicoccoides) lines from Israel and in particular the accession FA15-3 which is able to accumulate over 40% protein when given adequate nitrogen (Avivi, 1978) Joppa and Cantrell (1990) assigned the locus controlling this trait to chromosome 6B using substitution lines of emmer chromosomes into durum wheat cv Langdon and the precise gene has since been identified, mapped and designated Gpc-B1 (Distelfeld et al., 2004, 2006; Olmos et al., 2003) The QTL associated with this locus appears to account for about 70% of the variation in GPC in crosses (Chee et al., 2001; Joppa et al., 1997) However, Chee et al (2001) also showed a negative correlation with grain yield The FA15-3 line of emmer has also been used to transfer the Gpc-B1 gene into hard red spring wheats (Khan et al., 1989) Three lines were produced and contained up to 3% more protein than the parental lines Frohberg also transferred the Gpc-B1 gene into bread wheat to produce the only commercially released hard red spring cultivar with genes derived from emmer, called Glupro (quoted in Khan et al., 2000; Mesfin et al., 2000) RFLP markers to facilitate the transfer and selection of this locus have also been developed (Distelfeld et al., 2006; Khan et al., 2000; Mesfin et al., 1999) Mesfin et al (2000) carried out detailed comparisons of lines derived by single-seed descent from crosses between a low protein parent (Bergen, 14.5% protein) and two high protein parents derived from FA15-3 emmer (ND683 and Glupro, both approximately 18.5% protein) The grain yields of 12 high protein and 12 low protein lines from each cross did not differ significantly, indicating that it may be possible to combine high protein and high yield in commercial lines Recent work has shown that the Gpc-B1 gene encodes a transcription factor that accelerates senescence resulting in increased mobilisation and transfer of nitrogen and minerals (zinc, iron) to the developing grain (Uauy et al., 2006) Hence, lines expressing this allele contain higher amounts of iron and zinc in their grain as well as higher protein (Distelfeld et al., 2006; Uauy et al., 2006) Other studies have identified QTLs for grain protein on chromosomes 5A, 5D, 2D, 2B, 6A, 6B and 7A of bread wheat (Blanco et al., 2002; Groos et al., 2003; Snape et al., 1993; Turner et al., 2004; Worland and Snape, 2001) and ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 on chromosome 5B as well as 6B of emmer wheat (Gonzalez-Hernandez et al., 2004) Worland and Snape (2001) suggested that less than 30% of the variation in protein content of UK winter wheats can be explained by known genes and suggested that this applied to wheat in general The only major exception to this may be the Gpc-B1 QTL which can account for up to 70% of the variation in crosses Despite the work discussed above, there is no doubt that progress in breeding high protein wheats has been disappointing compared with that made with most other quality or agronomic traits This is partly due to the multigenic control and strong environmental impacts which make it difficult to select effectively However, a more important limitation is the inverse correlation between yield and protein content which is well established for all cereals (reviewed by Simmonds, 1995) Consequently, there is almost always a yield penalty when growing high protein cereals, which means that a higher price must be guaranteed to ensure support from farmers Although the theoretical basis for this inverse correlation has been debated (Miflin, 1980; Penning de Vries, 1975; Penning de Vries et al., 1974) the practical outcome is that high protein cereals are unlikely to be commercially successful without financial incentives to growers The reader is also referred to Konzac (1977) for an exhaustive account of early work on GPC in wheat Grain protein composition Protein nutritional quality is determined by the proportions of essential amino acids, which cannot be synthesised by animals and hence must be provided in the diet If only one of these amino acids is limiting, the others will be broken down and excreted resulting in poor growth of livestock and humans and loss of nitrogen in the diet Ten amino acids are strictly essential: lysine, isoleucine, leucine, phenylalanine, tyrosine, threonine, tryptophan, valine, histidine and methionine However, cysteine is often also included as it can only be synthesised from methionine (which is itself essential), with combined values for these sulphur-containing amino acids being presented Similarly, combined values for the biosynthetically related aromatic amino acids phenylalanine and tyrosine are often presented Finally, the requirements are different for adults where amino acids are required for maintenance and for children where they are also required for growth (Table 1) Higher values are also recommended for rapidly growing livestock, with the precise values depending on the growth rate The amino acid compositions of a range of cereals are given in Table These are from a range of sources and expressed as either g amino acid/100 g protein or g amino acid/16 g N (these units are regarded as being essentially the same, although the conversion factor for N to protein varies from about 5.1 to 6.0, depending on the cereal species and its nitrogen content (Mosse´, 1990)) Although these data are from a range of sources they clearly illustrate 241 several important points Firstly, although lysine is the limiting amino acid for all cereals, the amount varies between species, being highest in oats and rice and lowest in wheat and maize Similar differences also occur in the proportions of other essential amino acids with tryptophan being particularly deficient in maize Values are also given for whole grain (wholemeal) and starchy endosperm (white flour) fractions from wheat The latter shows a significant decline in the proportion of lysine, which is the first limiting amino acid The contents of amino acids in whole cereal grains are largely determined by the starchy endosperms which typically comprise about 80% of the grain dry weight The starchy endosperm is the major storage tissue of the grain, storing starch and proteins The latter are predominantly prolamins in all cereals except oats and rice in which the major storage proteins are related to the 11S globulins (‘‘legumins’’) of legumes and other dicotyledonous plants, with the prolamins being only minor components The essential amino acid compositions of these storage protein fractions (Table 2) show that the content of lysine is higher in the legumin-type storage proteins of oats and rice than in the prolamins of cereals, accounting for the higher contents of lysine in these cereals Similarly, tryptophan is absent from the major prolamin fraction (a-zeins) of maize accounting for the low content of this amino acid in the whole grain of this cereal The aleurone and embryo tissues of grains contain higher contents of essential amino acids (approximately 4.8 and 8.3 g% lysine, respectively, in wheat) (Jensen and Martens, 1983) but these are often not available for human nutrition as they are removed by milling (wheat), polishing (rice), pearling (barley) and decorticating (sorghum) Because the EAA composition of cereal grains is determined by their low levels in the prolamin storage proteins, the quality of the grain is influenced by factors which affect the proportions of these proteins in the grain For example, smaller grains will tend to contain higher proportions of EAAs in the whole grain of non-starchy endosperm tissues, but this will not affect the composition of polished grain or white flour The nutritional quality of the grain also decreases with increasing GPC, as an increasing proportion of the nitrogen is incorporated into prolamin storage proteins This is illustrated for barley by Fig 1A which shows data from field trails in the UK (Kirkman et al., 1982) Fig 1B shows that a similar trend occurs in other cereals, including oats in which the major globulin storage proteins are more lysine rich (Mosse´ and Huet, 1990) However, nitrogen application also increases cereal yields and the farmer must therefore balance the optimum nitrogen requirement for yield with the commercial requirements for either high or low protein grain 3.1 High lysine cereals In 1964 Mertz and co-workers reported that a naturally occurring mutant line of maize, called opaque-2, contained 69% more lysine than normal maize (Mertz et al., 1964) ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 242 Table Comparison of the contents of essential amino acids in cereal grain and flours with the FAO recommended levels for children and adults Wheat Histidine Isoleucine Leucine Lysine Cysteine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Barley Oats Rye Rice Maize Fao recommendations Grain White flour Grain Groat Grain Milled Cornflour Children Adults 2.3 3.7 6.8 2.8 2.3 1.2 4.7 1.7 2.9 (1.1) 4.4 2.2 3.6 6.7 2.2 2.5 1.3 4.8 1.5 2.6 (1.1) 4.1 2.3 3.7 7.0 3.5 2.3 1.7 5.2 2.9 3.6 1.9 4.9 2.2 3.9 7.4 4.2 1.6 2.5 5.3 3.1 3.3 ND 5.3 2.2 3.5 6.2 3.4 1.9 1.4 4.5 1.9 3.3 1.1 4.8 2.4 3.8 8.2 3.7 1.6 2.1 4.8 2.5 3.4 1.3 5.8 2.7 3.6 12.5 2.7 1.6 1.9 5.0 3.8 3.7 0.6 4.8 2.6 4.6 9.3 6.6 4.2 1.6 1.3 1.9 1.6 1.7 7.2 1.9 4.3 1.7 5.5 0.9 0.5 1.3 d d e e a a b c d Values are g/100 g protein or g/16 g N a Means of values for wholemeal and white flour samples of five types of wheat (hard red winter, hard red spring, soft red winter, club and durum) Calculated from data in Shoup et al (1966) Values for tryptophan are taken from single analyses reported in Paul and Southgate (1978) b Means of values for eight samples each of six-rowed and two-rowed barleys Calculated from data in Newman and McGuire (1985) c Means of values for 289 samples (Robbins et al., 1971) d Calculated from single analysis reported by Paul and Southgate (1978) e FAO/WHO/UNU (1985) Table Approximate abundances (% total grain N) and essential amino acid (EAA) compositions (mol%) of storage protein fractions from major cereal grains Prolamins Legumin-like Wheat Gliadin % grain N EAAs Histidine Isoleucine leucine Lysine Cysteine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Glutenin Barley Maize Oats Rice Oats Rice Hordein Zein Avenin Prolamins Globulin Glutelin 1–5 75 75–90 33 16 50 1.8 3.8 6.6 0.7 2.4 1.3 6.0 2.8 1.7 ND 4.2 0.9 3.4 6.6 1.2 1.3 1.3 5.4 3.2 2.5 ND 3.5 2.3 3.3 7.1 0.8 3.0 1.4 6.0 3.4 3.6 ND 4.7 a a b 52 10 1.0 3.8 18.7 0.1 1.0 0.9 5.2 3.5 3.0 3.6 c 0.9 3.4 10.8 0.9 3.8 2.0 5.5 1.6 1.7 ND 7.6 d 1.7 12.3 4.4 1.0 t 0.8 4.4 6.4 1.3 1.6 7.0 d 2.2 4.8 7.4 2.9 1.1 0.9 5.2 3.5 4.1 1.0 6.4 f 2.1 7.0 4.1 2.3 1.7 1.7 4.1 3.7 3.0 1.0 6.8 e a Data for gliadins and for glutenin fractions extracted with 70% ethanol and 0.05 N acetic acid (Dubetz et al., 1979) Tryptophan was not determined but Bushuk and Wrigley (1974) reported values of 0.7 and 2.2g/16 N, respectively, for similar fractions b Values for grain at weeks post anthesis (Shewry et al., 1979) c Protein % if for total zein in whole grain EAA composition is for the major azeins from six separate sources Tryptophan value is based on sequences of a-zeins derived from gene sequences Taken from Wilson (1987) d Kim et al (1978) e Protein % from Juliano (1985), EAA composition from Padhye and Salunkhe (1979) f Protein % from Colyer and Luthe (1984), EAA composition from Brinegar and Peterson (1982) This landmark report was followed by the identification of the floury-2 mutant which contained a similar amount of lysine to opaque-2 (Nelson et al., 1965) and by the discovery of a number of other high lysine maize mutants (Dalby and Tsai, 1975; Ma and Nelson, 1975; Misra et al., 1975; Salamini et al., 1979; Tsai et al., 1978) The high lysine mutants of maize were initially identified based on visual phenotypes and this approach was ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 therefore applied to sorghum However, only two high lysine lines were found amongst 62 floury lines identified in the world sorghum collection (9000 accessions), which both contained a recessive gene/s called hl (Singh and Axtell, 1973) A further high lysine mutant called P721 opaque was subsequently identified in a mutagenised population of the inbred line P721 (Axtell et al., 1979) Visual screening is not possible with barley so Munck et al (1970) used a direct screen for basic amino acids (dye binding capacity) to identify a single-high lysine line in 2500 accessions from the world barley collection As with sorghum, this discovery was followed by analysis of mutant populations, leading to the identification of almost 20 new mutations resulting from treatment with chemical (ethylinimine, sodium azide, ethylmethane sulphonate) or physical (g-rays, fast neutrons, thermal neutrons) mutagens (reviewed by Doll, 1983; Shewry et al., 1987) Although these mutant genes vary in their mechanisms most result in decreased synthesis of the lysine-poor prolamin fraction and compensatory increases in other 4.5 4.0 3.5 40 3.0 35 Lysine (mol %) Hordein N (% total N) 50 2.5 450 500 550 600 650 700 750 800 Total N (µg/seed) more lysine-rich fractions, which may include elevated levels of free lysine This is illustrated for maize in Table 3, which is taken from an excellent recent review of high lysine maize by Gibbon and Larkins (2005) However, the high lysine mutations in all cereals are associated with negative effects on yield while in maize and sorghum the softer grain may be less acceptable to some cultures and more prone to damage and infection A considerable amount of time, effort and money has been spent on trying to incorporate these genetic sources of high lysine into commercial varieties with acceptable yields and grain characteristics, with largely disappointing results In barley attention has been focused on two lines; Hiproly, which was the original high lysine mutant identified by Munck et al (1970) and Risø 1508, which was induced by treatment with ethylinimine (Ingversen et al., 1973) Breeders at the Carlsberg Research Laboratory focused on the lys3a gene from Risø 1508 and released a spring barley cultivar with improved lysine content for livestock feed (appropriately named Piggy) in 1987 However, this variety was not successful, presumably because the increased value for feed was not sufficient to offset the small yield penalty Studies of Hiproly also failed to result in the development of commercially successful varieties but did identify lysine-rich proteins (Hejgaard and Boisen, 1980) which were subsequently exploited in genetic engineering (as discussed below) Similarly, the high lysine P721 opaque line of sorghum continues to be used for research (see Oria et al., 2000) but has not been developed commercially In fact, it is perhaps appropriate that the only high lysine gene which has been successfully incorporated into commercial lines is opaque-2, which was the first to be discovered This has been achieved by using modifying genes to give ‘‘quality protein maize’’ 3.2 Quality protein maize (QPM) Oats Lysine (g/16g N) 243 Rice Barl So ey Wh ea Mai t ze rgh um Total N (g/100g dry wt.) Fig (A) The relationship between total grain nitrogen, % hordein (prolamin storage proteins) and % lysine in field grown grain of barley Taken from Kirkman et al (1982) with permission (B) The relationship between total seed N and lysine content Taken from Mosse´ and Huet (1990) with permission The development of QPM resulted from painstaking research carried out by S.K Vasil and E Villegas at CIMMYT (the international centre for the improvement of maize and wheat) in Mexico over two decades They selected for opaque-2 lines which contained increased lysine but had normal endosperm texture, which proved to result from suppression of starchy phenotypes by modifier (mo) genes (Prasanna et al., 2001) Biochemical analysis of these lines demonstrated that the decreased content of the 22-kDa a-zeins which occurs in opaque-2 mutants is accompanied by increases in another group of zein proteins, the 27-kDa g-zeins (Geetha et al., 1991; Lopes and Larkins, 1991), and it has been suggested that these proteins contribute to modification of the starchy phenotype by forming a cross-linked network around the starch granules (Dannenhoffer et al., 1995) QPM varieties have now been released in many countries in Latin America, Africa and Asia and are contributing significantly to increased nutritional quality for humans ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 244 Table Protein composition and lysine content of selected opaque mutants of maize Line Total protein (%) Zein protein (%) Non-zein protein (%) Non-protein N (%) Lys (%) Reference W64A+ W64Ao1 W64Ao2 W64Ao5 W64Ash4-o9 W64Ao11 W64AMc W64ADeB30 W64Afl2 A69Yfl1 A69Yfl3 BA hybrid 12.1 12.8 10.1 11.5 12.2 12 11.7 12 11.8 12.7 11.9 13.5 8.2 8.5 2.9 6.4 7.3 6.4 7.2 4.5 5.9 7.5 4.9 NA 2.1 2.1 3.6 2.8 3.4 2.7 3.8 3.7 ND ND ND 0.6 0.7 2.3 1.5 1.3 1.4 1.4 1.7 0.9 ND ND ND 1.5 1.7 3.8 2.1 2.8 2.1 2.9 2.8 4.1 3.9 a a a a a a a a a b b c Taken from Gibbon and Larkins (2005) with permission a Hunter et al (2002); b Balconi et al (1998); c Segal et al (2003) and livestock (Prasanna et al., 2001) However, it must be borne in mind that this has resulted from substantial investment over almost 40 years and that similar investment has not been available for other cereals One breeding company in the USA has also produced high-yielding hybrid opaque-2 varieties for production of feed grain in the USA, although these are not defined as QPM as they retain the soft endosperm texture (Crow and Kermicle, 2002) 3.3 High methionine maize Methionine is synthesised in plants from aspartic acid by the same biochemical pathway as two other essential amino acids, lysine and threonine Enzymes in this pathway are feedback inhibited by these two amino acids (Galili, 1995), meaning that maize embryos are unable to grow on medium supplemented with lysine and threonine due to starvation for methionine Phillips et al (1981) therefore screened 200 whole kernels of inbred maize lines for root growth on medium supplemented with lysine and threonine and identified a single line (BSSS53) which has an elevated content of grain methionine Subsequent studies showed that this line was enriched by 30–70% in the 10 kDa d-zein component (Phillips and McClure, 1985), which contains over 20 mol% lysine However, attempts to transfer the high methionine trait into other hybrids and inbreds failed with no significant increases in methionine being observed Genetic engineering approaches The failure of most attempts to improve grain nutritional quality by conventional approaches (including mutation breeding) has led to the use of genetic manipulation (GM), with several approaches being adopted 4.1 Increasing lysine-rich proteins Analysis of Hiproly, the spontaneous high lysine mutant of barley (Munck et al., 1970) revealed that the increased content of lysine resulted mainly from increases in four proteins: b-amylase (5.0 g% lysine), protein Z (a serpin proteinase inhibitor, 7.1 g% lysine) and two lysine-rich inhibitors of chymotrypsin called CI-1 (9.5 g% lysine) and CI-2 (11.5 g% lysine) (Hejgaard and Boisen, 1980) This result was important as it demonstrated that substantial increases in lysine-rich proteins could occur without any adverse effects on grain development CI-2 also has been used for a range of fundamental studies of protein structure and folding (see Clore et al., 1987; McPhalen and James, 1987; Otzen and Fersht, 1995, 1998) which have facilitated its exploitation as a basis for designing further lysine-rich forms for use in nutritional enhancement Roesler and Rao (1999, 2000) used CI-2 to design a range of nutritionally enhanced forms, one of which contained 11 lysine, five methionine, two tryptophan, one glycine and three threonine substitutions in the same protein Hence this form contained a total of 14 lysine residues out of a total of 83 residues The introduction of a single disulphide bond resulted in stability which was close to that of the wild type protein (which contains no cysteine residues), indicating that highly substituted forms of CI-2 may be suitable for expression in transgenic crops The highly substituted forms of CI-2 reported by Roesler and Rao (1999, 2000) have not yet been expressed in transgenic plants but a less substituted form is currently being used to transform grain sorghum (O’Kennedy et al., 2006) This contains three additional lysine residues substituted into the inhibitory loop region, resulting in 13.1 mol% lysine in the whole protein (Forsyth et al., 2005) Similar studies have been reported using another small lysine-rich protein from barley, hordothionin ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 Hordothionin contains five lysine residues out of a total of 45 residues but Rao et al (1994) have designed and tested mutant forms containing up to 27% lysine One form containing 12 lysine residues (HT12) has been expressed in grain sorghum, resulting in a 50% increase in total grain lysine (Zhao et al., 2003) The work discussed so far has exploited two proteins which are naturally present in cereal grain They may therefore be considered to be safe and acceptable by consumers and regulatory authorities In contrast, Yu et al (2004) expressed a lysine-rich protein which is normally expressed in potato pollen in grains of maize This protein contains 40 lysine residues out of 240 amino acids (16.7 mol%) and expression in maize resulted in increases in both grain protein and grain lysine (by up to 50%) However, the lack of information about the biological role and properties of this protein could well cause concern for use in food crops Finally, Wu et al (2003) reported an innovative approach to increase total grain lysine by substituting lysine residues for glutamine, asparagine and glutamic acid residues This was achieved by transforming rice to express tRNAlys species that introduce lysine instead of these other residues The substitutions are not specific for any proteins but the authors argued that their targeting to residues which are frequently exposed on the protein surface (glutamic acid, glutamine and asparagine) and their infrequent introduction would not lead to deleterious effects In the case of rice, the lysine content of the grain prolamins was increased by 43% and of the whole grain by 0.9% 4.2 Decreasing zein synthesis The decrease in zein synthesis which occurs in high lysine maize mutants results in increased grain lysine but there are also pleitropic effects on other grain characteristics including reduced yield Genetic engineering has therefore been used to specifically down-regulate zein synthesis, aiming to avoid the deleterious effects associated with the high lysine mutations Two reports have used RNA interference (RNAi) technology to specifically down-regulate the major 22 kDa (Segal et al., 2003) and 19 kDa (Huang et al., 2004) a-zeins in transgenic maize, respectively, reproducing the effects of high lysine genes However, the increases in grain lysine (by 15–20%) was substantially less than that in opaque-2 and other high lysine mutants The studies also confirmed that reduction in a-zeins was sufficient to result in an opaque phenotype although this was more pronounced when the 22 kDa a-zeins were suppressed More recently, Huang et al (2006) used a similar approach to simultaneously down-regulate the 19 and 22 kDa zeins in the same line The contents of grain lysine increased from 2,438 ppm dry weight in the wild type to 5,003, 4,533 and 4,800 ppm dry weight in three lines, while the contents of tryptophan also increased from 598 ppm 245 dry weight to 1,087, 940 and 1,040 ppm dry weight The seed size and protein content were not significantly affected and these increases corresponded to increases in lysine from 2.83% to 5.62% of the total protein and in tryptophan from 0.69% to 1.22% 4.3 Expression of high methionine proteins The high content of methionine in the 10 kDa d-zein and the association of this protein with high methionine content in line BSSS53 (discussed above) led to an attempt to over-express this protein using transgenesis (Kleese et al., 1991) Although some transgenic kernels contained 30% more methionine than normal grain, the relationship between the presence of the transgene, d-zein accumulation and methionine content was not clear cut More recent work showed that the expression of the 10 kDa d-zein gene (drz10) is regulated post-transcriptionally by a trans-acting factor encoded by the drz1 locus on a separate chromosome (Cruz-Alvarez et al., 1991; Schickler et al., 1993) To eliminate this regulation Lai and Messing (2002) produced transgenic maize plants in which the expression of the 10 kDa d-zein gene was driven by the promoter of the 27 kDa g-zein gene which lacks the site for posttranscriptional regulation by dsz1 Although only a single transgenic line was analysed this contained a similar level of methionine to BSSS53; approximately double that in one inbred line The transgenic line also gave a similar effect on weight gain in feeding tests with rats to supplementation with free methionine 2S albumin storage proteins are widespread in seeds of dicotyledenous plants although they are not present in cereals They include methionine-rich forms in some species (see Shewry and Pandya, 1999) and two of these have been expressed in transgenic rice as sources of high methionine Hagan et al (2003) used a gene encoding SFA8, an albumin from sunflower seeds which contains 16 methionine and eight cysteine residues in a mature protein of 103 residues (Kortt et al., 1991) Expression under the control of an endosperm-specific glutenin promoter from wheat resulted in an increase in total seed methionine but a decrease in cysteine of about 15% Hence, the main impact was on the distribution of sulphur within the grain rather than on the total sulphur content and there was little effect on the overall nutritional quality Similar effects had been reported previously when methionine-rich proteins were expressed in dicotyledenous seeds (Molvig et al., 1997; Tabe and Droux, 2002) suggesting that sulphur supply to the seed may limit attempts to increase the methionine content Two more recent studies have used a 2S albumin gene from sesame which encodes a protein with similar contents of methionine (15 residues) and cysteine (8 residues) to SFA8 (Tai et al., 1999) This gene was expressed in the bran (Lee et al., 2005) and starchy endosperm (Lee et al., 2003), using oleosin and glutelin promoters, respectively The transgenic seeds contained elevated contents of these amino ARTICLE IN PRESS 246 P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 acids: 24–38% more methionine and 50–62% more cysteine in the bran (Lee et al., 2005) and 29–76% more methionine and 31–75% more cysteine in the total seed proteins (Lee et al., 2003) However, neither of these studies provided detailed analyses of the content and distribution of sulphur within the seeds and it is not possible to conclude whether the overall nutritional quality was substantially improved Another important consideration when using 2S albumins as sources of increased methionine in transgenic cereals is that many proteins of this group are allergenic (Monsalve et al., 2004), including SFA8 of sunflower (Kelly and Hefle, 2000; Kelly et al., 2000) and sesame 2S albumin (Beyer et al., 2002; Wolff et al., 2003) which are discussed above There has also been the well-publicised case of the expression of the allergenic methionine-rich 2S albumin of Brazil nut in soybean (Melo et al., 1994; Nordlee et al., 1996) which resulted in the material being withdrawn from development Although this has been taken to indicate that GM technology is inherently unsafe it is in fact a clear indication of the effectiveness of current regulatory procedures Because of their potential allergenicity, it is unlikely that transgenic plants expressing any methionine-rich 2S albumins will be accepted by regulatory authorities for human consumption Corynebacterium in canola and a five-fold increase by transforming soybean with the same gene together with an AK gene from E coli (Falco et al., 1995) However, the situation is clearly less straightforward in maize Expression of the Corynebacterium DHDPS gene in the aleurone and embryo (under control of the globulin gene promoter) resulted in a substantial increase in total grain lysine, by 50–100% (Mazur et al., 1999) However, no increase in free lysine occurred when the same gene was expressed in the starchy endosperm, controlled by the glutelin (ie g-zein) promoter However, the breakdown product a-amino adipic acid did accumulate (see Fig 2), which suggests that increased lysine catabolism occurred Similar accumulation of a-amino adipic acid and saccharopine (also a degradation product) was also reported in transgenic canola and soybean (Falco et al., 1995; Mazur et al., 1999) Recent work on transgenic Arabidopsis has shown that this effect can be overcome by expressing the DHDPS gene in a knockout mutant lacking activity of lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) (Fig 2) (Zhu and Galili, 2003) The DHDPS transgenic and the knockout mutant produced 12-times and five-times the amount of free lysine present in the wild type line, respectively However, the combination of the transgene and knockout gave an 4.4 Increasing free amino acids The compensatory increases in other nitrogenous fractions which occur in a number of high lysine mutants include increases in free (i.e non-protein) amino acids (see Table for maize mutants) and this fraction has also been a target for genetic engineering studies Free amino acids usually account for low proportions of the total amino acids in the grain (see Table for maize) and only increase above these levels if the seed is restricted in its ability to synthesise proteins, for example, under conditions of sulphur limitation (Shewry et al., 1983) or in high lysine mutants (as discussed above) The low levels of free amino acids are maintained by complex regulatory networks in which end products of pathways inhibit enzymes controlling key steps in their biosynthesis The amino acids lysine, threonine and methionine are synthesised from aspartic acid by the pathway shown in Fig Their levels are controlled by feedback inhibition, with both lysine and threonine inhibiting the first enzyme in the pathway, aspartate kinase (AK) In addition, lysine and threonine also inhibit enzymes controlling the branch points leading to their synthesis, homoserine dehydrogenase (HDH) and dihydrodipicolinate synthase (DHDPS), respectively (reviewed by Galili, 1995, 2002) Attempts have therefore been made to increase the flux through this pathway by transforming plants to express feedback insensitive forms of AK and/or DHDPS, usually derived from bacteria (Escherichia coli or Corynebacterium) Thus, a two-fold increase in free lysine was acheived by expression of a feedback insensitive form of DHDPS from Fig Outline of the biosynthetic pathway leading from aspartic acid to lysine, threonine and methionine and of lysine catabolism Thick lines show enzymic steps with broken lines showing several steps Thin lines show feedback regulatory loops AK, aspartate kinase; HSD, homoserine dehydrogenase; DHDPS, dihydrodipicolinate synthase; LKR, lysine ketoglutarate reductase; SDH, saccharopine reductase ARTICLE IN PRESS P.R Shewry / Journal of Cereal Science 46 (2007) 239–250 80-fold increase in free lysine with free methionine also increasing by up to 38-fold Recently (September 2006) the Monsanto company released the first commercial GM high lysine corn, in which the high lysine trait is combined with herbicide resistance and resistance to the European corn borer This line expresses the feedback-insensitive DHDPS gene from Corynebacterium controlled by the maize globulin (Glb1) promoter This promoter targets expression to the germ and aleurone and the increase in free lysine is reported to result in increases in total grain lysine from about 2500–2800 ppm dry weight to about 3500–5300 ppm dry weight (Monsanto petition number 04-CR-114U) A similar approach can also be used to increase the content of free tryptophan in seeds Thus, expression in rice seeds of a mutated feedback-insensitive form of the endogenous biosynthetic enzyme anthranilate synthase resulted in increases in free tryptophan of between 55- and 431-fold (Wasaka et al., 2006) This corresponded to increases of between two- and 12-fold in the total tryptophan content Conclusion: GM approaches to nutritional improvement It is clear from the small number of studies discussed above that there is potential to use GM to improve grain nutritional quality However, none of the transgenic approaches discussed have so far been shown to produce lines which are competitive with conventionally bred cultivars in their yield and agronomic 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