Zein Accumulation Research Articles

Storage protein characteristics of proline-requiring mutants of Zea mays (L.)

Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro 1-1, pro 1-2, and pro 1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro 1-2 and pro 1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.

Zein level in Maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci

Zeins, the major endosperm proteins of maize, represent about 50% of total seed proteins and consist of several alcohol-soluble polypeptides encoded by at least four families of genes. The accumulation of zeins is under the control of several broadly defined regulatory genes. One of these, the opaque-2 mutant, lowers the level of zeins, thus improving the nutritional quality of maize meals. We demonstrate here that a salt-soluble protein of molecular weight 32,000 (b-32) is under control of O2. Seven o2 recessive alleles are CRM − when assayed with b-32 antibody. The O2 gene does not encode protein b-32, which apparently is the gene product of the O6 locus. o6 endosperms are CRM − for b-32 protein and are almost devoid of zeins. This indicates that b-32 plays an important role in accumulation of zeins.

Storage proteins in Zea mays (L.): interrelationship of albumins, globulins and zeins in the opaque-2 mutation.

Mature endosperms from opaque-2 mutant seeds with various genetic backgrounds (B 37, W 64A, R 802A and Oh 43) contained twice as much globulin than the corresponding normals, reduced amounts of zein and an increased amount of albumin. The latter is caused by a diminished disappearance of albumins in mutant endosperms during the final phases of development as compared to normal endosperms. Albumins from the opaque-2 endosperms appeared on gel electrophoresis as a similar but heterogenous polypeptide population comparable to that of normal endosperms except that in all opaque-2 forms a 70-kDa polypeptide was increased. Similarly, one to three specific globulin polypeptides (47 kDa, 52 kDa and 58 kDa) were, depending on the genotype, increased in the mutant lines. The accumulation of albumins, globulins and zeins, studied in developing W 64A opaque-2 mutant and corresponding normal maize kernels from 10 days after pollination until maturity, demonstrated two-phasic accumulation patterns for all proteins in the mutant, the first phase extending to about 30 days post pollination and the second one from there on until maturity. The transition time-point from first to second phase was characterized in mutant endosperm by a sudden reduction in accumulating albumins (also seen in normal endosperm), by an enhanced accumulation of globulins and the cessation of further accumulation of zein. Preferential accumulation of certain globulins has been found in the mutant during the second phase of globulin formation. The increased accumulation of globulins in the opaque-2 mutant endosperm is considered a response to the arrest in zein synthesis.

Amino Acid Composition of Grain Protein of Maize Grown with and without Pesticides and Standard Commercial Fertilizers1

AbstractIncreases in the price of agricultural chemicals and uncertain supplies are likely to cause a reduction of pesticide and fertilizer use in the future. Therefore, it is of interest to compare yields and quality of crops grown under present conventional practice with those grown at the lower limit of chemical intensiveness. In this paper, we report measurements of the amino acid composition of protein from maize (Zea mays L.) grain raised on 14 pairs of fields which did (conventional) and which did not (organic) receive pesticides and standard commercial fertilizers. The pairs of fields were matched for location, cultivar, planting date, and soil type.When expressed as percent of grain protein, five amino acids were at significantly higher concentration in organic grain and three were at significantly higher concentration in conventional grain. However, because of the lower protein concentration in the organic maize grain, most of the amino acids in conventional grain had significantly higher concentrations, when expressed as percent of total grain weight. The latter fact is more important from the standpoint of livestock feeding.Differences in yield, protein concentration, and amino acid composition between organic and conventional maize were qualitatively similar to reported differences between N‐fertilized and non‐N‐fertilized maize. At least part of the difference between organic and conventional maize is consistent with a greater accumulation of zein in conventionally raised maize.Results presented in this paper suggest that organic maize yields could be increased by N application. Since the yield difference was small, the N limitation should be overcome by modest rates of application.

A Proposed Role of Zein and Glutelin as N Sinks in Maize

Zea mays grown with high levels of N fertilizer transports more sucrose into kernels than with low N. Sucrose translocation was greatest in genotypes with the highest capacity to deposit nitrogenous compounds as zein and glutelin in the kernel. These two proteins combined contain about 80% of the total N in the kernel and about 60% of the total N in the plant at maturity. They appear to serve as a functional N sink for the deposition of nitrogenous compounds. As the N sink capacity increases with additional available N fertilizer, more sucrose is transported into the kernel, resulting in increased kernel weight and grain yield. Zein functions as a more dynamic N sink than glutelin because the synthesis of zein is readily manipulated by N fertilization and genetic means. Increases in N deposition in the normal endosperm induced by N fertilizer are confined primarily to zein. Early termination of zein accumulation in the opaque-2 mutant results in a reduction of sucrose movement into kernels. By using plants heterozygous for normal and opaque-2 in these studies, interplant variability was eliminated and the hypothesis relating the kernel N sink capacity to productivity was strengthened.

Effects of floury-2 locus on zein accumulation and RNA metabolism during maize endosperm development.

Zein accumulation patterns during mutant and normal maize endosperm development were determined. Accompanying an increase in the number of floury-2 alleles present in the endosperm was a well-defined stepwise depression in zein accumulation. Analysis of the zein accumulated in endosperms containing zero, one, two, and three doses of the floury-2 allele by sodium dodecylsulfate--polyacrylamide gel electrophoresis revealed a proportionate reduction in the two major zein components, Z1 and Z2. In contrast, the relative proportions of the minor zein bands were altered. Membrane-bound polysomes isolated from kernels of floury-2 and normal maize were predominantly large size classes. The presence of increasing numbers of the floury-2 allele in the endosperm decreased recovery of membrane-bound polysomal material in a stepwise fashion. However, major alterations in polysome size-class distributions were not observed. The reduction in membrane-bound polysome material correlated linearly with reductions in in vitro zein synthesis and in vivo zein accumulation.

Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize

The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.

Storage Protein Synthesis in 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.

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.