Trends in Genetics
Molecular genetic approaches to developing quality protein maize
Introduction
Cereals typically provide ∼50% of the dietary protein for humans and can comprise up to 70% of the protein intake for people in developing countries. The demand for cereal grains will continue to increase as a consequence of the expanding human population, which could add >1.5 billion people by the year 2025 [1]. However, cereals do not provide a nutritionally balanced source of protein. The most abundant proteins they contain, the prolamin storage proteins, are devoid of several amino acids that are essential for monogastric animals, lysine being most limiting. Most cereals contain 1.5–2% lysine, whereas 5% is required for optimal human nutrition [2].
The mature maize kernel contains an embryo and a much larger endosperm, both of which are surrounded by the seed coat (Figure 1a). In typical dent maize varieties, the endosperm consists of several distinct regions that have different physical properties. The outermost layer, the aleurone, is composed of specialized cells that secrete hydrolytic enzymes during germination. Beneath the aleurone are starchy endosperm cells that are filled with starch and storage proteins. These cells create two distinct regions, the so-called ‘vitreous’ or glassy endosperm and the ‘starchy’ endosperm. Vitreous endosperm transmits light, whereas the starchy endosperm does not. Typically, the endosperm is ∼90% starch and 10% protein. Nearly 70% of the protein is composed of several types of prolamin proteins known as zeins (Figure 1b; Box 1). These form insoluble accretions called protein bodies in the lumen of the rough endoplasmic reticulum (ER), and during kernel maturation, the protein bodies become densely packed between starch grains in the vitreous regions of the endosperm.
The discovery that the opaque2 (o2) mutation increases the lysine content in maize endosperm by decreasing the synthesis of zein proteins and increasing the level of other lysine-rich proteins indicated that such mutants might be useful for improving human food and animal feed (Box 2). However, the low seed density and soft texture of this type of mutant were associated with several inferior agronomic traits, including brittleness and insect susceptibility. With only a few exceptions, these mutants were not commercially developed [3]. But not long after the discovery of o2, maize breeders began to identify modifier genes that alter the soft, starchy texture of the endosperm, giving it a normal appearance while maintaining the increased essential amino acid content of o2 (Figure 1c). The loci controlling this trait, ‘o2 modifiers’ (mo2) of which there are several, proved to be genetically complex but nevertheless effective in ameliorating the negative features of the opaque kernel phenotype. By systematically introgressing mo2 genes into o2 germplasm, plant breeders in South Africa and the International Maize and Wheat Improvement Centre (CIMMYT) in Mexico developed hard endosperm o2 mutants that they designated ‘Quality Protein Maize’ (QPM) 4, 5. QPM has the phenotype and yield potential of normal maize, but it maintains the increased lysine content of o2.
The development and widespread use of QPM germplasm has been slow, partly because of the technical complexity of introducing multiple mo2 loci, while maintaining a homozygous o2 locus and monitoring the amino acid composition. This process could be greatly accelerated if we understood the basis for the increased lysine content of o2 mutants and the mechanism(s) by which mo2 genes create a hard, vitreous endosperm. Consequently, recent research has attempted to determine the molecular basis for these two traits.
Section snippets
Cloning and characterization of opaque mutants
Maize mutants with uniform starchy endosperms generally fall into three classes: the recessive mutants opaque1 through opaque17 (o1,o2, o5, o7, o9–o11, o13–o17), the semi-dominant floury mutants (fl1, fl2 and fl3), the dominant mutants Mucronate (Mc), and Defective endosperm B30 (De-B30). Figure 2 shows the position of these mutations and zein genes on the maize genetic map. Studies suggest that the recessive mutations affect regulatory genes and that the semi-dominant and dominant mutations
opaque2 modifying genes
Because of the poor pest resistance and processing characteristics of starchy endosperm mutants, breeders worked to identify genotypes that restore the vitreous endosperm phenotype in o2 backgrounds (Box 2). The most effective o2 modifier genes provided the background for QPM [28]; these modifiers effectively suppress the starchy o2 phenotype, with little loss of protein quality. Inheritance of these modifier genes is complex and is likely to involve several loci [4], which complicates the
Relationship of starch structure to endosperm modification
A proteomic analysis to examine non-zein proteins that contribute to the vitreous phenotype of QPM was reported for nearly isogenic lines of CM105+, o2 and mo2 [39]. This study revealed a prominent change in a cluster of 56-kDa polypeptides that was increased by approximately tenfold in CM105mo2. These polypeptides were identified by peptide-mass mapping as granule-bound starch synthase I (GBSS I), the product of Waxy1. GBSS I is increased in the non-zein fraction of all QPM varieties tested so
Future prospects
The mechanisms by which the increased level of 27-kDa γ-zein and altered starch structure in QPM lead to more vitreous endosperm are unknown. However, given that γ-zein is present on the exterior surface of protein bodies, it is likely that as ER membranes disintegrate during desiccation, γ-zein can come into direct contact with starch granules. Zeins are found to be the major protein associated with the periphery of the starch granules in the mature seed [41]. Therefore, it is possible that
Concluding remarks
Recent research on improved protein quality and modification of kernel texture in QPM has created the opportunity for rapid progress in understanding the mechanisms underlying these traits. Important missing information includes the discovery of specific genes that control these phenotypes. One vital approach to better understand these mechanisms is identification of QTLs that are associated with protein quality and with the hard endosperm phenotype. Molecular markers have been identified that
Acknowledgements
We thank Rudolf Jung, Paolo Sabelli and Brenda Hunter for helpful comments on this article, and João Leiva-Neto for providing the illustration of a maize kernel. Research in the Larkins laboratory is supported by grants from the Department of Energy (DE-FG02–96ER20242), USDA-NRI (2004–00918) and Pioneer Hi-Bred International.
References (47)
Regulation of lysine catabolism in higher plants
Trends Plant Sci.
(2000)- et al.
A trip to the ER: coping with stress
Trends Cell Biol.
(2004) - et al.
The biochemical basis and implications of grain strength in sorghum and maize
J. Cereal Sci.
(1999) The end of world population growth
Nature
(2001)Significance of dietary protein source in human nutrition: animal and/or plant proteins
- et al.
Oliver Nelson and quality protein maize
Genetics
(2002) Quality protein maize
Curr. Sci.
(2001)Maize: the long trail to QPM
Maize opaque endosperm mutations create extensive changes in patterns of gene expression
Plant Cell
(2002)A new opaque variant of maize by a single dominant RNA-interference-inducing transgene
Genetics
(2003)