Endosperm Protein Quality and Kernel Modification in the Quality Protein Maize Inbred Lines

Twenty-two selected quality protein maize (QPM) lines, including 13 lines developed in India (DMRQPM series) and nine lines released by CIMMYT, Mexico (CML series), were evaluated for their endosperm protein content and quality, besides kernel modification in terms of vitreousness. Endosperm protein contents in 13QPMlines were on par or better than that of the normal maize ‘checks’ (Trishulata and Parkash). The QPM endosperm proteins showed significantly higher % tryptophan as well as EF-1α (a multifunctional protein with a positive and highly significant correlation with lysine content in the endosperm) contents, in comparison with the normal maize genotypes. Evaluation of kernel modification revealed considerable scope for accumulation of endosperm modifiers in some of the QPM lines. Positive and highly significant correlation was revealed between tryptophan and EF-1α contents in the endosperm proteins, whereas the correlations between the quality parameters with kernel modification in the QPM genotypes were found to be non-significant. The study led to the identification of some promising QPM lines, such as DMRQPM-37, DMRQPM-44, CML176, CML142 and CML149, which could be effectively deployed in the QPM breeding programmes.     

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.     

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque2 gene in the cross V25×CML176, optimum population size for the BC₂ generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC₂ generation identified three plants with 93 to 96% recurrent parent genome content. The three BC₂F₂ families derived from marker identified BC₂ individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25–50% and more than 50% opaqueness. BC₂F₃ lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germplasm.     

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.     

Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize

The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms.     

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.     

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.     

优质蛋白玉米02基因控制赖氨酸超量积累的分析

以３８个ＱＰＭ（或０２）和对照普通玉米为实验材料,进行０２基因控制赖氨酸超量积累的生化和遗传分析。主要实验结果如下：（１）ＱＰＭ玉米０２基因为隐性的单基因遗传,它控制着胚乳、雄穗和幼苗期叶片中赖氨酸的超量积累,一些修饰因子和遗传背景对胚乳物理性状产生影响;（２）ＱＰＭ玉米、普通玉米的胚较之胚乳,或者ＱＰＭ玉米胚乳较之普通玉米胚乳都含有较多的天门冬氮酸、甘氨酸、赖氨酸和精氨酸,含有较少的脯氨酸、谷氨酸、亮氨酸和苯丙氨酸;（３）两种玉米之间,在胚乳蛋白质含量及胚乳可溶性蛋白、醇溶蛋白、谷蛋白的赖氨酸含量方面没有什么不同;（４）已经育成一批ＱＰＭ或０２玉米自交系,并配制出几个强优势杂交组合。     

Some genetic and biochemical characteristics of a new source of maize opaque-2 mutant

Summary The opaque kernels separated from the F₁ of crosses of our opaque-2 strains with normal inbred lines contained 45, 50 and 74 percent more lysine in the whole kernel than the translucent kernels from the same ear. The opaque kernels from these crosses contained almost the same level of lysine as the parental opaque-2 strains.Some of our opaque-2 strains contain 63 to 122 percent more lysine than the tested dent or flint normal inbred lines. In comparison with opaque-2 Purdue, four opaque-2 strains had almost the same lysine content and a strain labelled as SP-1 No. 15 contained 25 percent more lysine and 73 percent more tryptophan. Some of our opaque-2 strains contained 55 to 100 percent more tryptophan in the whole kernel than opaque-2 Purdue.In contrast to lysine content, the transmission of the tryptophan content from opaque-2 strains to opaque kernels from their crosses shows variability.We found weak positive correlation (r = +0,3057) between lysine and tryptophan content in opaque-2 kernels.Our opaque-2 strains had a higher lysine content in the endosperm than did opaque-2 Purdue 22.1 – 36.6% more, and compared with normal lines they had 109.5 – 142.6% more.It is apparent that a new source of opaque-2 mutant gene, which has the same genetic and biochemical characteristics as the opaque-2 mutant discovered by Mertz, Bates and Nelson (1964), has been found in a completely new, genetically divergent, strain derived from a large number of populations.

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Zusammenfassung Die vorgenommenen Prüfungen haben gezeigt, daß undurchsichtige Körner, die nach F₁-Kreuzung unserer Opaque 2-Linien mit Normallinien ausgelesen wurden, 45, 50 und sogar 74% mehr Lysin im ganzen Korn als normale durchsichtige Körner desselben Maiskolbens enthalten. Der Lysingehalt der undurchsichtigen Körner aus diesen Kreuzungen entspricht annähernd dem der Opaque 2-Elternlinien.Einige unserer Opaque 2-Linien enthalten 63 bis 122% mehr Lysin als die untersuchten normalen Inzuchtlinien. Im Vergleich zu Opaque 2 — Purdue weisen unsere 4 Opaque 2-Linien einen etwa gleichen Lysingehalt auf, während die SP-1 No. 15 25% mehr Lysin und 73% Tryptophan enthält. Unsere Opaque 2-Linien zeigen im ganzen Korn 55 bis 100% mehr Tryptophan als Opaque 2 — Purdue.Im Unterschied zum Lysingehalt zeigt die Übertragung des Tryptophangehalts von Opaque 2-Linien auf undurchsichtige Körner bei den Kreuzungen Variabilität.Wir fanden eine schwach positive Korrelation (r = + 0,3057) zwischen dem Lysin- und Tryptophangehalt bei den Opaque 2-Köinern.Unsere Opaque 2-Linien hatten mehr Lysin im Endosperm als Opaque 2-Purdue, und zwar 22,1 bis 36,6% bzw. 109,5 bis 142,6% im Vergleich zu den normalen Linien.Offensichtlich ist eine neue Quelle für das mutante Opaque 2-Gen in einer ganz neuen, genetisch unterschiedlichen Synthese einer großen Anzahl von Populationen entdeckt worden. Die neue Quelle hat dieselben genetischen und biochemischen Merkmale wie die Opaque 2-Mutante, die von Mertz, Bates und Nelson (1964) aufgefunden wurde.

     

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     

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.     

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.     

Genetic analysis of amino acid accumulation in opaque-2 maize endosperm

The opaque-2 mutation in maize (Zea mays) is associated with an increased level of free amino acids (FAA) in the mature endosperm. In particular, there is a high concentration of lysine, the most limiting essential amino acid. To investigate the basis for the high-FAA phenotype of opaque-2 maize, we characterized amino acid accumulation during endosperm development of several wild-type and opaque-2 inbreds. Oh545o2 was found to have an exceptionally high level of FAA, in particular those derived from aspartate (Asp) and intermediates of glycolysis. The FAA content in Oh545o2 is 12 times greater than its wild-type counterpart, and three and 10 times greater than in Oh51Ao2 and W64Ao2, respectively. We crossed Oh545o2 to Oh51Ao2 and analyzed the F(2:3) progeny to identify genetic loci linked with the high FAA level in these mutants. Quantitative trait locus mapping identified four significant loci that account for about 46% of the phenotypic variance. One locus on the long arm of chromosome 2 is coincident with genes encoding a monofunctional Asp kinase 2 and a bifunctional Asp kinase-homo-Ser dehydrogenase-2, whereas another locus on the short arm of chromosome 3 is linked with a cytosolic triose phosphate isomerase 4. The results suggest an alternation of amino acid and carbon metabolism leads to overproduction and accumulation of FAA in opaque-2 mutants.     

Genetic analysis of opaque2 modifier loci in quality protein maize

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.     

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     

Expression of alcohol-soluble endosperm proteins in maize single and double mutants

Summary Many maize (Zea mays L.) mutant genes exist. Some affect protein content or composition, while others modify carbohydrates or kernel phenotype. In doublemutant lines, two mutant genes are present. We know little about interactions of such genes, however. We therefore examined a normal maize inbred, B37, 10 near-isogenic single mutants and 46 double mutants to analyze quantitative effects on alcohol-soluble endosperm proteins. Proteins were extracted with 70% ethanol0.5% sodium acetate-5% mercaptoethanol, and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Early peaks were alcohol-soluble glutelin (ASG) subunits, while late peaks contained zein. Results were quantified and statistically analyzed. In many double mutants, protein compositions differed significantly from averages of compositions of corresponding single mutants. For example, a high-methionine, water-insoluble ASG is absent when the opaque-2 (o2) gene combines with shrunken-1 (sh1) or surgary-1 (su1). Another water-insoluble ASG nearly doubled when floury-2 (fl2) andsu1 combined. A high-proline, high-histidine, water-soluble ASG nearly doubled in combinations offl2 witho2,su1 and sugary-2 (su2). Zein was about half its expected value wheno2 combined with amylose-extender (ae), floury-1 (fl1), soft-starch (h),sh1 andsu1. Thus, rapid protein extraction and quantitative RP-HPLC showed major new epistatic and synergistic effects of several mutant genes on protein composition. Unexpectedly, these effects often involve genes that primarily affect starch composition or kernel phenotype. Alcohol-soluble proteins often vary in amount, as ino2 lines. They also differ in nutritional value. Thus, RP-HPLC analysis of these proteins can identify nutritionally superior genotypes, and may help explain the basis of such quality.Presented at the XVI International Congress of Genetics, Toronto, Canada, August 20–27, 1988     

Transaminase GOT and GPT activity in extirped sprouts of normal and opaque-2 Maize (Zea mays L.) seedlings

The increased activity of GOT (E.C.2.6.1.1.) and GPT (E.C.2.6.1.2.) transaminases in maize seedlings found as a marker of genotype opaque-2, was investigated in extirped sprouts of both genotypes, normal and opaque-2. The enzymatic activity was determined in three maize samples from breeding experiments, each sample consisting of a genotype pair, normal and opaque-2, collected from segregating ears of maize plants in the S₁ generation. The seedlings were aseptically grown for 7 days in two variants of cultivation, intact seedlings and sprouts extirped after 4 days of germination. In the intact seedlings of genotype opaque-2 an increased activity of GOT and GPT, as compared to the intact normal plants, was observed. The extirpation of the sprouts enhanced GOT and GPT activity in the sprouts of both genotypes. However, in extirped sprouts the GOT and GPT activity was found to be still higher in the genotype opaque-2 as compared with the sprouts of normal genotype. Thus it seems that the increased transaminase activity in the sprouts of genotype opaque-2 is genetically determined. The increase does not result from an induction of enzyme synthesis through the supply of amino acids translocated from the endosperm to the sprouts. The absolute level of transaminases in the different breeding samples is dependent on the parenteral lines, the relative level of GOT and GPT activities is higher in the genotype opaque-2.