Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.     

Influence of genotype and texture on zein content in endosperm of maize grains

The effect of genotypes and texture on the content of proteins in maize grains was examined by assessing absolute amounts of six protein fractions in the whole endosperms of four wild‐type lines with high protein content and four quality protein maize (QPM) varieties and for hand‐dissected hard and soft endosperm regions from eight other lines. As previously reported for six wild‐type lines and their opaque‐2(o2) versions, zeins were predominant for all genetic backgrounds and all types of endosperms. From these data and others the amounts of zeins and true proteins (crude proteins free of non‐protein nitrogen) in developing and mature endosperms of wild‐type lines were correlated. The data points for zeins from hard endosperms lay between the regression line and the upper limit of confidence area. Those for zeins from soft endosperms were located at the lower part of confidence area and on a level with the points corresponding to the most immature endosperms. Furthermore, some data points for zeins from o2 and QPM samples lay near the lower limit while the others were outside the confidence area. This suggested an initial zein accumulation dependent on the genotype at a low relative rate, followed by an accumulation at higher rate. The conditions used for isolating and quantitating zeins are discussed.     

A study of the sites of glycosylation of zein proteins using the lectin concanavalin A

The sites of glycosylation of zeins, the maize (Zea mays L.) storage proteins, were studied using the affinity of the lectin Concanavalin A (Con A) for certain glycosides. Zeins which were extracted from kernels of Illinois High Protein (IHP), W22, W64A and Oh43 were separated by isoelectric focusing and analyzed with a radiolabeled Con A binding technique. Certain sub-groups of the zein proteins contained carbohydrate moities which bound Con A while others did not. Zeins extracted from Oh43 kernels had a higher relative affinity for Con A than those of other maize lines. Further analyses of the zeins of Oh43 by gas chromatography demonstrated the presence of fucose, mannose and glucose.     

优质蛋白玉米胚乳贮存蛋白积累的电泳分析

玉米胚乳的２２ ｋＤ和２０ ｋＤ醇溶蛋白在授粉后１５ 天开始积累,编码２２ ｋＤ 和２０ ｋＤ醇溶蛋白的结构基因在胚乳发育过程中同时表达。优质蛋白玉米和ｏ２ 玉米的胚乳中,２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成受到抑制,即ｏ２ 基因对２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成有负的调节作用。Ｍｏ１７／ｏ２ 和Ｍｏ１７ 胚乳醇溶蛋白的双向电泳结果表明,Ｍｏ１７／ｏ２ 的２７ ｋＤ、２２ ｋＤ、２０ ＫＤ和１５ ｋＤ醇溶蛋白的合成均受到强烈的抑制。遗系０４１／ｏ２ 和遗系０４０／ｏ２ 胚乳醇溶蛋白的双向电泳结果表明,二者只在高分子量的蛋白质斑点区域有一些细微的差别。可溶性蛋白的ＳＤＳ－ＰＡＧＥ分析表明,Ｍｏ１７／ｏ２ 胚乳的可溶性蛋白比其同型系Ｍｏ１７ 少３８．７ ｋＤ 和２６．７ ｋＤ两条谱带,多２７．２ ｋＤ和２６．１ ｋＤ两条谱带。二者出现的可溶性蛋白的差异是ｏ２ 基因调控的结果。遗系０４１／ｏ２ 胚乳的可溶性蛋白比其同型系０４０／ｏ２ 多１８．６ ｋＤ和１７．６ ｋＤ两条谱带,少４０．２ ｋＤ 一条谱带,这与ｏ２ 基因修饰因子的作用密切关联     

Electromicrographic Analysis Storage Protein Accumulation in Endosperms of Maize with Qualified Protein

The characteristics of storage protein accumulation of maize with qualified protein (MQP) and 02 maize were analysed basing on the genetical and biochemical point of views. The 22 kD and 20 kD zeins in the developing endosperms of maize accumulated 15 days after pollination. The structural genes encoding 22 kD and 20 kD zeins in the developing endosperms were simutaneously expressed. In the endosperms of MPQ and o2 maize the synthesis of 22 kD and 20 kD zeins was suppressed. That is to say, o2 gene negatively regulated the synthesis of 22 kD and 20 kD zeins. Two-dimentional electrophoretic analysis of zeins in the maize endosperms further revealed the effects of o2 gene and its modifiers on the synthesis of zeins. In Mol7 and Mo17/o2 endosperms the synthesis of 27 kD, 22 kD, 20 kD and 15 kD zeins was severely suppressed. In 041/oz and 040/o2 endosperms little difference existed SDS-PAGE analysis of the soluble proteins of Mol 7 and Mo17/o2 endosperms revealted that two bands with molecular weight (MW) of 38.7 kD and 26.7 kD were present in wild type but absent in o2 mutant, while two bands with MW 27.2 kD and 26.1 kD were present in o2 mutant but absent in wild type. These differences were resulted from the effect of o2 gene. In 040/02 and 041/o2 endosperms two bands with MW 18.6 kD and 17.6 kD were present in 041/o2 but absent in 040/02 while one band with MW 40. 2 kD was present in 040/02 and absent in 041/o2, which was closely related to the effects of the modifiers of o2 gene.     

Rapid and simple method for the quantitative extraction of corn endosperm proteins

Rapid quantitative extraction of corn endosperm proteins is accomplished by presolubilization of the zeins in 70% ethanol ($\text{v}\text{v}$), followed by addition of dilute NaOH solution to the ethanolic slurry. The effectiveness of this technique rests primarily on the rapid solubilization of the zeins in weakly ethanolic dilute NaOH solution and on the prescribed sequence of addition of reagents. The extraction conditions of endosperms of vitreous and floury phenotypes differ as to sample particle size and optimum concentrations of NaOH.     

Expression of A/B zeins in single and double maize endosperm mutants

Summary Zeins, the major endosperm proteins in maize (Zea mays L.), are deficient in the essential amino acids lysine and tryptophan. Some mutant genes, like opaque-2 (o2) and floury-2 (fl2), reduce the levels of A- and B-zeins, thereby improving maize's nutritional value. Other mutants, such as amylose-extender (ae), floury-1 (fl1), soft starch (h), dull-1 (du), shrunken-1 (sh1), sugary-1 (su1), sugary-2 (su2), and waxy (wx), primarily affect starch composition, but also alter zein composition. We undertook this study to examine the effects of some of these mutant genes on A/B-zein composition and to study the interactions of these genes in double-mutant combinations. Endosperm prolamins were extracted from inbred B37, ten near-isogenic single mutants (ae, du, fl1, fl2, h, o2, sh1, su1, su2, and wx), and most double-mutant combinations. Zeins in these extracts were fractionated by reversed-phase highperformance liquid chromatography (RP-HPLC) into 22–24 peaks. Of the resulting 22 major peaks the areas of 16 (per milligram endosperm) were significantly affected by individual mutant genes relative to the zein composition of the normal inbred. In combination these genes exhibited significant epistatic interactions in regulating the expression of individual A/B zeins. Epistatic interactions were judged to be significant when the amount of a peak in a double mutant differed from the averages for the peak in the two respective single mutants. The o2 gene, alone and in combination with other mutant genes, significantly decreased the amounts of many individual zeins. The effect of the o2 gene was the greatest of all the genes examined. Various clustering techniques were used to see if mutant effects could be grouped; among these was principal component analysis, a multivariate statistical technique that analyzes all peak sizes simultaneously. Three-dimensional scatter graphs were constructed based on the first three principal components. For the single mutants, these showed no relationships to gene actions; for the double mutants, however, this technique showed that four single mutants, o2, sh1, su1 and su2, had the greatest effects on zein composition when combined with each other and with the remaining six single mutants.Presented at the XVI International Congress of Genetics, Toronto, Canada, August 20–27, 1988. The mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA over other brands or similar products not mentioned     

Genetic control of a membrane component and zein deposition in maize endosperm

Summary An association is reported between an albuminlike protein (b-70) and the semidominant locus fluory-2 (fl2) which reduces the level of zein polypeptides in the maize endosperm. The protein b-70 is present at low level in wild-type endosperms and derppressed in fl2 endosperms. A correlation between the doses of the fl2 allele and the b-70 level has been found. Moreover a concomitant loss of the regulatory role of fl2 on zein level and on b-70 overproduction is evident when fl2 is genetically associated with o2 and o7, two recessive alleles of other zein regulatory loci. Protein b-70 is located on the membrane of the protein body where zein polypeptides accumulate. The existence of a functional relationship between this protein and the zein-secretory system is suggested or, as an alternative, that b-70 is a type of storage protein different from zeins, repressed in normal endosperms and derepressed by the fl2 allele.Abbreviations DAP days after pollination - ER endoplasmic reticulum - RER rough endoplasmic reticulum - DTT dithiothreitol - EDTA ethylene-diamintetra-acetate - NADH nicotinamide-adenine dinucleotide, reduced - PMSF phenylmethylsulfonyl-fluoride - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.15 M NaCl, 0.01 M Na phosphate, pH 6.8)     

Induction of tetraploid derivatives of maize inbred lines by nitrous oxide gas treatment

Maize (Zea mays L.) is a model organism for various genetic and physiological studies. Induction of autotetraploid lines from elite inbred lines is valuable for investigating gene dosage effects on the molecular level. We applied nitrous oxide gas at the time of fertilization (30-36 h after pollination) for 20 h on maize inbred line Oh43. The nitrous oxide gas treatment between pressures of 600-1000 kPa proved to be effective in inducing tetraploids. The treatment also significantly increased the rates of germless and shriveled kernels. Twelve inbred lines were treated with nitrous oxide gas for 20 h at either 800 or 900 kPa pressures, 30 or 36 h after pollination. Although tetraploid or tetraploid class aneuploid plants from 9 of 12 inbreds tested were successfully generated, only six genotypes produced progenies. The successful tetraploid inbred lines were from the A188, B73, H99, Oh43, Stock 6, and W22 genetic backgrounds. Aneuploids, plants with broken chromosomes and chimeras, were also found among the treated materials.     

Relationship between structural and biochemical characteristics and texture of corn grains

The texture of corn grains is a fundamental characteristic for the industry as well as for grain producers and processors. To further understand the mechanisms involved in grain hardness, contrasting corn cultivars for grain hardness and protein quality were evaluated. The cultivars were Cateto L237/67 (hard endosperm and low protein value), QPM BR 451 (semi-hard endosperm and high protein value); Bolivia-2 (floury endosperm and low protein value), and Opaque-2 (floury endosperm and high protein value). Evaluations were carried out at 10, 20, 30, 40, 50, and 60 days after pollination in developing corn grains and in the mature grain. In developing grains, evaluation consisted in the determination of the area, percentage of starch granules, distribution of starch granules, and protein bodies in the endosperm. In mature corn grains, the parameters evaluated were: density, percentage of total proteins, levels of lysine and tryptophan, and banding pattern of zeins. The results indicate that the higher physical resistance of corn grains from the cultivars analyzed is influenced by the high percentage of total proteins, high synthesis of 27-kDa zeins, presence of protein bodies, and perfect organization of starch granules in the endosperm, independent of their sizes.     

Organ-specific invertase deficiency in the primary root of an inbred maize line

An organ-specific invertase deficiency affecting only the primary root system is described in the Oh 43 inbred maize (Zea mays). Invertases (acid and neutral/soluble and insoluble) were assayed in various tissues of hybrid (NK 508) and inbred (Oh 43, W 22) maize lines to determine the basis for an early report that Oh 43 root tips were unable to grow on sucrose agar (27). Substantial acid invertase activity (7.3 to 16.1 micromoles of glucose per milligram of protein per hour) was evident in extracts of all tissues tested except the primary root system of Oh 43. This deficiency was also evident in lateral roots arising from the primary root. In contrast, morphologically identical lateral roots from the adventitious root system had normal invertase levels. These results suggest that ontogenetic origin of root tissues is an important determinant of invertase expression in maize. Adventitious roots (including the seminals) arise above the scutellar node and are, therefore, of shoot origin. The Oh 43 deficiency also demonstrated that invertase activity was not essential for maize root growth. Sucrose synthase was active in extracts from all root apices and theoretically provided the only available avenue for sucrose degradation in primary root tips of Oh 43. The deficiency described here will provide a useful avenue of investigation into the expression and significance of root invertase.     

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。     

Genotypic Variation for Glycinebetaine among Public Inbreds of Maize

Screening of a range of public maize (Zea mays L.) inbred lines for glycinebetaine (betaine) content over two growing seasons (1987 and 1988), using fast atom bombardment mass spectrometry, has identified 19 public inbred lines which all exhibit low betaine levels (<100 nanomoles per gram fresh weight). These include common inbreds such as A188, A619, B37, H95, N6, and Oh43. Several inbreds exhibit high betaine levels (3000 to 10000 nanomoles per gram fresh weight); in these strongly betaine-positive inbreds, betaine levels tended to be, on average, 1.38-fold greater in the 1988 growing season presumably in part due to field water deficits experienced during the drought of 1988. Where several different sources of the same inbred line were available (including cytoplasmic male sterile and restored lines of A632, B37, B73, Oh43, and WF9), betaine levels were found to be similar when the inbreds were tested in the same environment. Because W22-R/r-X1 was found to be strongly betaine-positive, it should be possible to map the putative recessive gene(s) determining betaine deficiency to specific chromosome(s) from monosomics resulting from crosses between W22-R/r-X1 and betaine-deficient lines.     

The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ～1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.     

Multivariate analysis of polypeptide synthesis in field-grown maize inbreds and hybrids

Summary A leaf disc method is described to permit the localized incorporation of ³⁵S-l-methionine into polypeptides synthesized in individual leaves of maize plants grown in the field. The method of incorporation employs minimal external manipulation of the intact leaf, is simple, repeatable, and may be used at any plant age after leaf emergence. Incorporation (cpm/g protein) in 12 leaves per plant was compared among three inbred (Oh43, W23, M14) and three F₁ hybrid (Oh43/M14, W23/M14, Oh43/W23) genotypes. The incorporation was 40% higher (hybrid versus inbred) in 9 of the 12 leaves studied. Samples from leaf 07 (7th leaf numbered from base of plant) for four inbreds (Oh43, M14, B73, Mol 7) and two pairs of reciprocal F₁ hybrids (Oh43/M14, M14/Oh43; B73/Mo17, Mo17/B73) were labelled in situ using the leaf disc method. Each cultivar was sampled at three different ages in each of 1985, 1986, and 1987. High-resolution, two-dimensional isoelectric focusing sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and fluorography were used to display the polypeptides synthesized in the samples. Multivariate methods — Principal Coordinate Analysis, Cluster Analysis, and Standard Deviation Distance — were used to analyze variation and to identify trends in the variation for year, genotype, and age sampled. Our analyses disclose a hierarchy to polypeptide synthesis variation in maize leaves: differences in polypeptide synthesis are greater for year-to-year comparisons than differences due to sample age, which in turn are greater than differences for inbred versus hybrid comparisons.     

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.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.