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.     

Characterization of polysomes and incorporation in vitro of leucine and lysine in normal and opaque-2 zea mays endosperm during development

Polysome preparations obtained from opaque-2 and normal maize endosperms during development did not show any significant difference in sedimentation coefficient or nucleotide composition. The pattern of incorporation in vitro of lysine and leucine, however, differed quite distinctly in these two preparations. During early stages of maturity the polysomes from opaque-2 incorporated substantially more lysine and less leucine as compared with those from normal maize. Although the trend was reversed at 25 days post-pollination, this did not result in any significant zein accumulation since very little total protein was synthesized after that stage in opaque-2 maize endosperm. It is, therefore, suggested that the opaque-2 gene exerts a regulatory control on mRNA synthesis, required for zein formation at early stages of maturation.     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Genetic regulation of storage protein content in maize endosperm

Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals that zein prepared from normal maize inbred (Zea mays L.) contains six separable components. Z1 and Z2 are the predominant species, with molecular weights of 21,800 and 19,000 daltons. Amino acid analysis of these two components shows that both are rich in glutamic acid, leucine, and proline, but low in lysine. Of the four minor bands, Z3, Z4, Z5, and Z6, the latter two exist only in trace amounts. A mutation at the opaque-2 locus severely suppresses the synthesis of Z1. The nonallelic mutant, opaque-7, strongly suppresses the synthesis of Z3 and Z4, while slightly reducing Z2. On the other hand, the floury-2 mutant appears to reduce the synthesis of these six proteins in the same relative proportion. In the double mutant combinations, opaque-2 apparently is epistatic to opaque-7 and floury-2 in the synthesis of zein components. The glutelin fraction shows a more complex banding pattern; however, qualitative differences are not apparent among the mutant lines examined.This research was supported in part by a grant from the Lilly Endowment.Journal Paper No. 6100 of the Purdue University Agricultural Experimental Station.     

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.     

Adenine methylation in zein genes

This paper reports the novel finding of adenine methylation in higher plants. Comparison of restriction patterns of genomic maize DNA digested with enzymes MboI and Sau3A enabled us to detect the existence of adenine methylation in zein genes. Adenine methylation within or around zein genes turned out to be similar in endosperm (where zeins are actively synthesized) and in seedling tissue (where zein genes are not expressed). Furthermore, adenine methylation patterns were found to be similar both in wild-type and opaque-2 mutant plants. These lines of evidence suggest that adenine methylation is unrelated to the regulation of gene expression.     

Ribonuclease Activity in Normal, opaque-2, and floury-2 Maize Endosperm during Development

The elevated ribonuclease activity produced in the endosperm of a maize (Zea mays L.) inbred, W64A, by homozygous opaque-2, results from a more than doubled rate of ribonuclease accumulation occurring prior to 16 days post-pollination; after 16 days the rates in opaque-2 and normal are the same, suggesting that opaque-2 is no longer active. The pattern of ribonuclease increase in the opaque-2 dosage series indicates that opaque-2 is not fully recessive. Ribonuclease accumulation is not affected by floury-2 in a second inbred, B14. The results are discussed with reference to other proteins, notably zein, the net synthesis of which is affected by opaque-2.     

Chromosomal localization of zein genes by in situ hybridization in Zea mays

Summary In order to localize the genes coding for zein, the major storage protein of maize endosperm, zein ¹²⁵I-mRNA and ³H-cDNA labelled at high specific activity were used for in situ hybridization on heterozygous interchanges and paracentric inversions of the KYS strain of Zea mays. The analysis of the diplotene-metaphase I microsporocytes indicated the presence of zein structural genes on the long arm of chromosomes 4 and 5, the short arm of chromosome 7 and the distal segment of the long arm of chromosome 10. The two hybridization sites on chromosomes 7 and 10 are found near opaque-2 and opaque-7 loci which are known to regulate zein synthesis. The present data are discussed in relation to results obtained by other authors using genetical mapping of zein genes.     

Tissue-specific zein synthesis in maize kernel

Zein may account for as much as 10% of the total protein in the mature embryo of maize inbred W64A. This protein exhibited an electrophoretic pattern on SDS gels similar to that of the endosperm. Like the endosperm system, the synthesis of zein components in the embryo was controlled by the opaque-2 and floury-2 mutations. However, unlike zein synthesis in the endosperm, zein synthesis in the embryo could not be increased by nitrogen fertilizer. Variations in amino acid composition were observed between the zein components of the embryo and those of the endosperm.     

Additive and Non-additive Genetic Correlations. II. Application to Data Obtained from a 12 × 12 Diallel Cross

The proposed genetic correlation analysis, that involves the partitioning of the overall genetic correlation into an additive and a non-additive component, has been applied to data obtained from a diallel experiment involving 12 white modified opaque-2 maize inbred lines. The correlation analysis provided an insight into possible indirect selection strategies for the improvement of inferior kernel quality traits associated with the opaque-2 gene. Direct selection for high yield and low vitreousness rating would provide an efficient selection strategy for the development of high-yielding modified opaque-2 maize hybrids with desirable endosperm traits. It was concluded that it is not necessary to conduct the density, hardness and breakability determinations.     

Identification of opaque-2 genotypes in segregating populations of Quality Protein Maize by analysis of restriction fragment length polymorphisms

Quality Protein Maize (QPM) is a name given to genetically modified opaque-2 maize with hard endosperm. The opaque-2 mutation conditions a reduction in the amount of zein seed storage protein; zeins are deficient in the essential amino acids lysine and tryptophan, and mutant seed have a higher nutritional value. To utilize the potential of opaque-2 maize, elite inbreds can be converted to o2/o2 forms and subsequently to hard endosperm opaque-2. Since opaque-2 is recessive and endosperm specific, conventional backcross procedures to convert elite inbreds to opaque-2 forms are inefficient. To alleviate this problem, a marker-assisted selection procedure was developed for the Texas A&M University Quality Protein Maize breeding program. Hybridization of an O2 cDNA probe to blots of DNA from plants carrying O2 and o2 alleles showed that restriction fragment length polymorphisms (RFLPs) exist between the W64A o2 allele and O2 alleles of Mo17 and TX5855 inbred lines. To identify the opaque2 genotypes in segregating populations, an RFLP marker assay combining the O2 cDNA probe and HindIII-digestion of genomic DNA was developed. The effectiveness of the O2 RFLP marker assay was tested under field conditions using F₂ and backcross populations of several hard endosperm opaque-2 lines. A comparison of the genotypes identified by RFLP analysis with the seed phenotypes of the next generation indicated that this procedure is accurate and can be used for identifying O2/O2, O2/o2, and o2/o2 genotypes of individual juvenile plants in breeding populations.     

Interaction of the opaque-2 gene with starch-forming mutant genes on the synthesis of zein in maize endosperm

The combination of opaque-2 with starch-modified or starch-deficient mutants produced a cumulative and synergistic effect, respectively, in regulating zein synthesis. The double mutant, brittle-2 opaque-2, which almost completely prevented the synthesis of Z1 and Z2, had high RNase activity. The possible involvement of RNase in effecting zein synthesis is discussed.     

Temporal profiling of essential amino acids in developing maize kernel of normal,opaque - 2 and QPM germplasm

Maize, an important cereal crop, has a poor quality of endosperm protein due to the deficiency of essential amino acids, especially lysine and tryptophan. Discovery of mutants such as opaque-2 led to the development of nutritionally improved maize with a higher concentration of lysine and tryptophan. However, the pleiotropic effects associated with opaque-2 mutants necessitated the development of nutritionally improved hard kernel genotype, the present-day quality protein maize (QPM). The aim of present study was to analyze and compare the temporal profile of lysine and tryptophan in the developing maize kernel of normal, opaque-2 and QPM lines. A declining trend in protein along with tryptophan and lysine content was observed with increasing kernel maturity in the experimental genotypes. However, opaque-2 retained the maximum concentration of lysine (3.43) and tryptophan (1.09) at maturity as compared to QPM (lysine-3.05, tryptophan-0.99) and normal (lysine-1.99, tryptophan-0.45) lines. Opaque-2 mutation affects protein quality but has no effect on protein quantity. All maize types are nutritionally rich at early stages of kernel development indicating that early harvest for cattle feed would ensure a higher intake of lysine and tryptophan. Two promising lines (CML44 and HKI 1105) can be used for breeding high value corn for cattle feed or human food in order to fill the protein inadequacy gap. Variation in lysine and tryptophan content within QPM lines revealed that differential expression of endosperm modifiers with varying genetic background significantly affects nutritional quality, indicating that identification of alleles affecting amino acid composition can further facilitate QPM breeding program.     

Heterogeneity of storage proteins in maize

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6–9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3–5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7–8) it has been calculated that at least 90% of the glutamic and aspartic acids (52 residues out of a total of 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.Abbreviations A absorbance - Bis N,N-methylene bisacrylamide - IEF isoelectric focusing - 2-ME 2-meroaptoethanol - mol wt molecular weight - 62 opaque-2 - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - PAS periodic acid-Schiff stain - SDS sodium dodecyl sulphate - ICA trichloroacetic acid - TEMED N,N,N,N-tetramethyl ethylene diamine - Z₁ zein extracted with 55% isopropanol - Z₂ zein extracted with 55% isopropanol and 0.6% 2-ME - Z 9.6 zein of mol wt 9600 - Z 13.5 zein of mol wt 13,500 - Z 21 zein of mol wt 21,000 - Z 23 zein of mol wt 23,000