Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm.

Grains of nine opaque (o) and floury (fl) mutants of maize (Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non-opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within (o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms.

Comparative efficiencies of isopropyl and tert-butyl alcohols for extracting zeins from maize endosperm.

Protein of endosperm of maize grains originating from three wild-type inbreds and their opaque-2 versions were solubilized in diverse extracts (E) by the sequential use of 0.5 M NaCl, water (E(1,2)), alcohol plus a reducing agent (E(3)), and salt plus a reducing agent (E(4)). Zeins were isolated in extracts E(3) and E(4) obtained by using 55% (w/w) isopropyl alcohol (i-PrOH) + 0.2% dithiothreitol (DTT) followed by 0.5 M NaCl + 0.2% DTT buffered at pH 10 or 60% tert-butyl alcohol (t-BuOH) + 0.2% DTT followed by 0.5% sodium acetate + 0.2% DTT in 30% t-BuOH. For a given genotype the percentage of extracted zeins was independent of the nature of the alcohol. The latter had a slight effect on the respective magnitude of E(3) and E(4): E(3) increased at the expense of E(4) when t-BuOH was substituted to i-PrOH for their isolation. The percentage of the total endosperm nitrogen present in E(3) + E(4) was identical to that of fractions F(II) + F(III) + F(IV) isolated according to the classical Landry-Moureaux extraction procedure. SDS-PAGE analysis revealed the presence of all types of zeins (alpha, beta, gamma, and delta) in E(3) and F(III), residual zeins in E(4) isolated with t-BuOH, and streaking only in E(4) and F(IV) isolated with NaCl at pH 10. The data together with those of the literature were discussed with regard to the influence of procedure on the yield of zeins using alcoholic extraction.

Regulation of lysine metabolism and endosperm protein synthesis by the opaque-5 and opaque-7 maize mutations.

Two high lysine maize endosperm mutations, opaque-5 (o5) and opaque-7 (o7), were biochemically characterized for endosperm protein synthesis and lysine metabolism in immature seeds. Albumins, globulins, and glutelins, which have a high content of lysine, were shown to be increased in the mutants, whereas zeins, which contain trace concentrations of lysine, were reduced in relation to the wild-type lines B77xB79+ and B37+. These alterations in the storage protein fraction distribution possibly explain the increased concentration of lysine in the two mutants. Using two-dimensional polyacrylamide gel electrophoresis of proteins of mature grains, variable amounts of zein polypeptides were detected and considerable differences were noted between the four lines studied. The analysis of the enzymes involved in lysine metabolism indicated that both mutants have reduced lysine catabolism when compared to their respective wild types, thus allowing more lysine to be available for storage protein synthesis.

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.

Genetic control of lysine metabolism in maize endosperm mutants.

The capacity of three maize endosperm opaque mutants (o10, o11 and o13) to accumulate soluble lysine in the seed in relation to their wildtype counterpart, W22+, was investigated. The W22o13 and W22o11 mutants exhibited 278% and 186% increases in soluble lysine, respectively, while for W22o10, a 36% decrease was observed, compared with the wildtype. A quantitative and qualitative study of the N constituents of the endosperm has been conducted and data obtained for the total protein, non-protein N, soluble amino acids, albumins / globulins, zeins and glutelins present in the seed of the mutants. Following 2D-PAGE, a total of 38 different forms of zein polypeptides were detected and considerable differences were noted between the three mutant lines. The metabolism of lysine was also studied by analysis of the enzymes aspartate kinase, homoserine dehydrogenase, lysine 2-oxoglutarate reductase and saccharopine dehydrogenase, which exhibited major changes in activity, depending on the genotype, suggesting that the mutant genes may have distinct regulatory activities.