Total Zein Research Articles

A linear model for quantitating the accumulation of zeins and their fractions (α+δ, β&γ) in developing endosperm of wild-type and mutant maizes

A model based upon the occurrence of a linear relationship between the amounts of zeins and total proteins present in developing maize endosperm was applied to some data of the literature. Those were concerned with grains of W64A, Illinois high and low protein (IHP and ILP) cultured in vivo and in vitro together with in vivo grown grains of opaque-2 ( o2) and Mucronate ( Mc) mutants, and Mco2 double mutant. The relative rate of zein accumulation, corresponding to the slope of a straight line, was found to be insensitive to environmental factors. It was assessed to 0.217, 0.431, 0.490, 0.598 and 0.799 for Mco2, ILP, o2, W64A and IHP maizes, respectively. The same type of relationship was tested between the amounts of zein fractions (α+δ, β and γ) and total zeins present in developing endosperm of three genotypes. The relative accumulation rate for a given fraction was very close to its proportion determined from mature endosperm irrespective of the genotype. According to the sign of y-intercept γ-zeins were found to be synthesized prior to (α+δ)-zeins, in agreement with the analysis of protein bodies using immuno-localisation; β-zein was synthesized after (α+δ)-zeins, contrary to the findings of immuno-localisation experiments, suggesting two different accumulation rates for this fraction.

Grouping and Identification of Argentine Maize Races by Principal Component Analysis of Zein Reversed‐Phase HPLC Data

ABSTRACTRacial classification of maize has important taxonomic and phylogenetic implications. It may also serve to organize germ plasm inventory, thus helping breeders choose their stocks. Maize racial classification is usually based on phenotypic descriptors, which may not always accurately express genetic characteristics. In contrast, synthesis and expression of zeins is directly associated with genotype. This study was conducted to determine whether racial grouping and identification of maize can be done by applying principal component analysis to zein reversed‐phase high‐performance liquid chromatography (RP‐HPLC) data. Zeins from samples of 97 landraces (primitive varieties) of the Argentine races Cristalino Colorado, Dentado Blanco, Avatí Morotí, Capia, and Pisingallo, stored at the Pergamino Active Maize Germplasm Bank, were analyzed by RP‐HPLC. Data from the ≈21‐ to 53‐min chromatogram region (total zeins [ZT]), the ≈21‐ to‐30 min region (zeins 2 [Z2]), or the ≈38‐ to 52‐ min region (zeins 1 [Z1]) were subjected to multivariate analysis based on principal component to group samples by race and to assign unknown samples to predetermined racial groups. Clearly differentiated racial groups were revealed, closely matching groups based on phenotype. Unknown samples could be assigned, with a low percentage of misidentification, to predetermined groups based on Mahalanobis distances. The shortest distances of unknown samples were almost always the distances to their respective groups. Approaches other than multivariate analysis were used to group and assign samples to defined races but they were not as effective. Results indicate the potential of this method as a complementary tool to perform racial grouping and identify maize materials with high genetic variability.

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

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 μm. 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 μm 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.

Analysis of zeins' ER retention in Xenopus oocytes

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.

Characterization of Polypeptides Corresponding to Clones of Maize Zein mRNAS

Zeins, the storage proteins of maize (Zea mays) are a complex group of polypeptides encoded by a large multigene family. The alpha-zein proteins, which account for about 70% of the total, show both size and charge heterogeneity. Although clones corresponding to several different alpha zeins have been characterized, it has not been possible to correlate these sequences with individual zein polypeptides. By translating in Xenopus oocytes RNAs transcribed in vitro from cloned zein mRNAs, we were able to identify the encoded proteins among native zeins or zeins synthesized in oocytes with total zein mRNA. There was no correlation between the isoelectric points of these proteins and the homology of their coding DNA sequences, as the proteins encoded by two closely homologous cDNAs migrated with greater charge heterogeneity than those encoded by less homologous clones. In addition, the size of the proteins as determined by SDS polyacrylamide gel electrophoresis did not always correlate with the length of the protein deduced from the DNA sequence. The ability to match cloned zein sequences to individual native proteins will enable the genetic mapping of cloned genes as well as the analysis of their translational regulation.

A proposed nomenclature for the alcohol-soluble proteins (zeins) of maize ( Zea mays L.)

A nomenclature system for maize (Zea mays L.) prolamins, zeins, is proposed. According to this system, the protein that is rich in proline, glutamine, leucine and/or alanine, and is extracted with aqueous alcohol solutions containing a reducing agent, is considered to be zein. Zein is separated into three distinct classes (α, β and γ) based on differences in solubility, amino acid composition, electrophoretic, chromatographic, and immunological properties, N-terminal amino sequences and complete amino acid sequences deduced from nucleic acid sequences. α-Zein constitutes 75–85% of the total zein and is made up of Mr-21,000–25,000 polypeptides plus a Mr-10,000 minor polypeptide. β-Zein includes Mrr-17,000–18,000 methionine-rich polypeptides and constitutes 10–15% of the total zein. γ-Zein is made up of one size class, Mr-27,000 proline-rich polypeptide, that constitutes 5–10% of the total zein.

Chromatographic and electrophoretic analysis of zein heterogeneity

Zein, purified from maize inbred W64A, was chromatographed on a column of hydroxypropylated Sephadex G-100 equilibrated with 75% ethanol containing 0·01 M HCI. The fractions obtained were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or acid-urea before and after reduction of disulfide bonds. Mr 22,000 polypeptides, free of Mr 24,000 polypeptides, were isolated from the proteins that were eluted last and were compared with total zein by two-dimensional electrophoresis consisting of isoelectric focusing in the first dimension and acid-urea gel electrophoresis in the second. The Mr 22,000 fraction consisted of at least nineteen proteins exhibiting microheterogeneity. These data indicate that zein comprises two sets of closely related polypeptides that exist free or as oligomers, mainly dimers.

Serial Analysis of Zein by Isoelectric Focusing and Sodium Dodecyl Sulfate Gel Electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.

Separation of Alcohol-Soluble Proteins (Zeins) from Maize into Three Fractions by Differential Solubility

The prolamin of maize (Zea mays L.), zein, was extracted from endosperm meal with 60% (v/v) 2-propanol/1% (v/v) 2-mercaptoethanol either directly or subsequent to extraction with 90% (v/v) 2-propanol. The zein extracted with 90% 2-propanol was essentially made up of 20 to 24 kilodalton polypeptides (alpha-zein) while that extractable with 60% 2-propanol/1% 2-mercaptoethanol contained, in addition to alpha-zein, 17 to 18 kilodalton methionine-rich polypeptides and a 27 kilodalton proline-rich polypeptide. While zein was separated into three fractions by differential solubility in 90% 2-propanol and 30% 2-propanol/30 millimolar sodium acetate (pH 6) using two different fractionation protocols. Each of the three solubility fractions (SF1, SF2, and SF3) had a unique polypeptide composition. Based on results obtained from two inbreds, K55 and W64A, the SF1 constituted 75 to 80% of the total zein and included as major components 20 to 24 kilodalton polypeptides and a minor 10 kilodalton polypeptide. The SF2 made up 10 to 15% of the total zein and included exclusively 17 to 18 kD methionine-rich polypeptides. A 27 kilodalton proline-rich component constituted the SF3 and contributed 5 to 10% to total zein.

Das Molekulargewicht des Zeins und sein Verhalten bei der Äthanol-Gradientextraktion in verschiedenen Reifestadien der Samen

Total zein from endosperms of maize, sampled at intervals after pollination, were subjected to pore-gradient electrophoresis and ethanol coneentration gradient analysis. Electrophoresis was conducted in the presence of SDS or urea. At different stages of maturity total zein is composed of the same 8 subunits. Subunits are associated in the same 12 aggregates. Quantitative interrelation between subunits and between aggregates is not changed during maturation of the seeds.

Storage Protein Synthesis in 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.

Amino acid composition of zein molecular components

Zein extracted from maize endosperm has been fractionated into four polypeptide chains, having the following MWs 23 000, 21 000, 13 500 and 9600. By amino acid analysis the two smaller MW chains (representing 30% of total zeins) have been found to be zein-type molecules. These two chains are thought to be responsible for zein granule formation via -S-S- bridges. Zein is also highly heterogeneous in charge, and is resolved into at least 15 components, with pI's in the pH range 5–9. As demonstrated by amino acid analysis, part of this heterogeneity is due to spot mutations in some of the genes responsible for zein synthesis.