High Lysine and High Tryptophan Transgenic Maize Resulting from the Reduction of Both 19- and 22-kD α-zeins

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD α-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, asparate and glutamate, was observed in the zein reduction kernels.     

Influence of opaque-2 and floury-2 genes on formation of proteins in particulates of corn endosperm

Protein-rich subcellular particulates were isolated by zonal centrifugation from homogenates of endosperms of normal, opaque-2, and floury-2 mutant corn (Zea maize) kernels at different stages of development. In early stages the high lysine mutants vary from normal corn by greater production of a glutelin protein not associated with the matrix. This protein is high in lysine and may become a component of matrix glutelin at later stages of maturity. Differences in size and structure of zein-rich protein bodies were observed in the mutant strains when compared with normal corn. Enhanced production of nonmatrix glutelin as well as the reduction in synthesis of lysine-deficient zein is responsible for the improved lysine content of the mutant endosperms at early stages of development.     

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize (Zea mays) mutants, brittle-2 (bt2), brittle-1 (bt), and shrunken-2 (sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

A proposed role of zein and glutelin as N sinks in maize

Zea mays grown with high levels of N fertilizer transports more sucrose into kernels than with low N. Sucrose translocation was greatest in genotypes with the highest capacity to deposit nitrogenous compounds as zein and glutelin in the kernel. These two proteins combined contain about 80% of the total N in the kernel and about 60% of the total N in the plant at maturity. They appear to serve as a functional N sink for the deposition of nitrogenous compounds. As the N sink capacity increases with additional available N fertilizer, more sucrose is transported into the kernel, resulting in increased kernel weight and grain yield. Zein functions as a more dynamic N sink than glutelin because the synthesis of zein is readily manipulated by N fertilization and genetic means. Increases in N deposition in the normal endosperm induced by N fertilizer are confined primarily to zein. Early termination of zein accumulation in the opaque-2 mutant results in a reduction of sucrose movement into kernels. By using plants heterozygous for normal and opaque-2 in these studies, interplant variability was eliminated and the hypothesis relating the kernel N sink capacity to productivity was strengthened.     

Synthesis of an unusual α-zein protein is correlated with the phenotypic effects of the floury2 mutation in maize

The soft, starchy endosperm of the maize (Zea mays L)floury2 mutant is associated with a reduction in zein mRNA and protein synthesis, unique protein body morphology, and enhanced levels of a 70 kDa protein, that has been shown to be the maize homolog of a chaperonin found in the endoplasmic reticulum. We found an unusual α-zein protein of 24 kDa to be consistently associated with the zein fraction from floury2 mutants. Three additional α-zein proteins with molecular weights ranging from ca. 25 to 27 kDa are detected in the storage protein fraction of a high percentage of floury2 kernels and a low percentage of normal kernels in a genetically segregating population. The four proteins can be distinguished from one another by immunostaining on Western blots. Synthesis of the 24 kDa protein is regulated by Opaque2, since the 24 kDa protein is lacking in the storage protein fraction of opaque2/floury2 double mutants. The synthesis of an abnormal a-zein protein in floury2 could explain many features of the mutant, such as the abnormal protein body morphology, induction of the 70 kDa chaperonin, and hypostasis to opaque2 (o2). Although we cannot prove that the accumulation of this protein is responsible for the floury2 phenotype, we were able to detect a restriction fragment length polymorphism (RFLP) linked to the floury2 locus with a 22 kDa α-zein probe. We hypothesize that the unique characteristics of the floury2 mutant could be a response to the accumulation of a defective a-zein protein which impairs secretory protein synthesis.     

In vitro synthesis of zein-like protein by maize polyribosomes.

Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes $\text{in vitro}$ produced one main radioactive protein. This protein was soluble in 70% ethanol and had the same mobility in electrophoresis on sodium dodecyl sulfate-gels as a zein standard. The ratio of [¹⁴C] leucine to [¹⁴C] lysine incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membrane-bound polyribosomes.     

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F₂ and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.     

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F₁ hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T₆ transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.