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.     

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.     

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.     

Genomic Organization of α-Amylase Gene in High Lysine Opaque-2 Maize

Genomic DNA was isolated from 7-day old etiolated seedlings of normal and high lysine opaque-2 maize and purified by CsCl gradient. Purified DNA was found to be ～48.5 kb in size. Restriction analysis of genomic DNA with EcoRI and HindIII did not reveal any noticeable difference between normal and opaque-2 DNA. Southern blot analysis, using α-amylase probe, showed multiple bands. One of the hybridizing bands (～4 kb) of genomic DNA was more intense in opaque-2 than in normal. This DNA was cloned into pUC 18 plasmid and presence of α-amylase was confirmed by Southern hybridization using α-amylase probe.     

New Methods for Extraction and Quantitation of Zeins Reveal a High Content of gamma-Zein in Modified opaque-2 Maize

We have developed methods for quantitative extraction and analysis of zeins from maize (Zea mays L.) flour. Extraction involved solubilization of total endosperm proteins in an alkaline buffer containing SDS and 2-mercaptoethanol with subsequent precipitation of nonzein proteins by the addition of ethanol to 70%. Analysis of these proteins by SDS-PAGE with Coomassie blue staining and by Western blotting and ELISA assay with zein antibodies revealed that this extraction method is more quantitative than the traditional Landry-Moureaux procedure, especially for the β- and γ-zeins. This method was used to extract and analyze the zein content of several `Quality Protein Maize' (QPM) varieties developed by the International Maize and Wheat Improvement Center. QPM varieties contain `modifier genes' that confer a vitreous phenotype on opaque-2 genotypes, while maintaining the elevated levels of lysine and tryptophan characteristic of this mutant. This analysis revealed that the QPM types contain 2 to 4 times the amount of the γ-zein than unmodified opaque-2 or normal maize varieties. Possible relationships between the high expression of the γ-zein and the modified opaque phenotype are discussed.     

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.     

Protein and free amino acids in a high lysine maize double mutant

A comparative study of free amino acids and protein fractions of normal with a double mutant (su₁ o₂) was made, during endosperm development in segregating ears of a maize synthetic. Zein content showed striking differences in the two genotypes, being 7.7 and 6 times greater in the normal endosperm at 24 and 47 days after pollination respectively. This observed decrease in zein synthesis, coded by sugary-1/opaque-2 genes, causes an accumulation of alanine, glutamic and aspartic acids, glutamine and asparagine in the high lysine endosperm mutant.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

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.     

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.     

Protein synthesis and changes in nucleic acids during grain development of Sorghum

Changes in DNA, RNA, nitrogen, nucleotide composition and in vitro incorporation of leucine/lysine by polysomes have been studied during sorghum grain development. Both DNA, RNA and protein content increased substantially during grain development. Although RNase activity increased, it did not affect RNA accumulation. Minor changes in the nucleotide composition of rRNA and sRNA were observed during grain development. In vitro incorporation of leucine and lysine by polysomes indicate qualitative change in the mRNA during later stages of grain development and the substantial accumulation of proteins during this period ultimately results in accumulation of proteins rich in leucine and poor in lysine.     

Evidence for the genetic control of lysine catabolism in maize endosperm

A split pollination was used to produce normal (Su su su O2 o2 o2) and high lysine double mutant sugary opaque-2 (su su su o2 o2 o2) endosperms on the same ear of sugary opaque-2 maize plants. Amino acids were determined in the vascular sap of the ear peduncle. Lysine content in the sap was compared with lysine stored in both normal and sugary opaque-2 endosperm during kernel filling. Lysine content in the ear peduncle sap could account for all lysine found in both endosperms. Preformed lysine is highly catabolized in the normal endosperm, but not in the high lysine sugary opaque-2 endosperm. The rate of lysine breakdown appears to be an important mechanism by which the high lysine mutant controls lysine level in maize endosperm.     

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.     

Changes in soluble protein and isoenzymes in normal and opaque-2 Zea mays endosperm during grain development

Electrophoretic patterns of soluble proteins, pH 5 enzyme fraction, peroxidase, glutamic dehydrogenase, leucine aminopeptidase in developing endosperm of normal and opaque-2 were studied. Multiple forms were found for all the enzymes studied. The GDH pattern showed considerable differences in normal and opaque-2 maize; the soluble protein pattern also differed, both qualitatively and quantitatively. The leucine-amino-peptidase pattern was identical and the peroxidase pattern showed slight differences.     

Storage Protein Synthesis in Maize: III. Developmental Changes in Membrane-bound Polyribosome Composition and in Vitro Protein Synthesis of Normal and Opaque-2 Maize

Zein synthesis accompanied an increase in large polyribosomes of maize (Zea mays) endosperm cells. The two classes of polyribosomes (free and membrane-bound) had dissimilar size class distributions. Membrane-bound polyribosomes were predominantly large size classes, which were not found in free polyribosomes. The ratio of large membrane-bound polysomes to total membrane-bound polysomes was highest when zein was being synthesized. Appearance of the large polysomes correlated with the onset of zein accumulation in vivo. These large size classes were nearly absent in the opaque-2 mutant at all stages of endosperm development. Similarly, rRNA content was reduced in the mutant from that in normal endosperm development. These differences were associated with reduced in vitro synthesis and in vivo accumulation of zein.     

A novel tubular array associated with protein bodies in the rough endoplasmic reticulum ofopaque-2 maize

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l m. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 m in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology