Zein Polypeptides Research Articles

Complex coacervation of zein-chitosan via atmospheric cold plasma treatment: Improvement of encapsulation efficiency and dispersion stability

Zein-polysaccharide complex coacervations were modified using atmospheric cold plasma (ACP) technology in order to decrease zein's hydrophobicity and consequent aggregation. The ACP-induced interaction between zein and chitosan in 70% ethanol-water solution was investigated. To evaluate the application potential of the modified complex in nutrition delivery, the encapsulation efficiency and dispersion stability of resveratrol-loaded nanoparticles were investigated. It was found that the novel zein-chitosan complex coacervation re-assembled in 70% ethanol-water solution after ACP treatment, displaying smaller particle sizes (from 1191 nm to 370 nm) and higher conductivity (from 36.6 ds/cm to 43.1 ds/cm) than non-ACP-treated samples. UV–vis spectrum, IR, AFM and SDS-PAGE analysis demonstrated that the ACP treatment caused conformational changes and unfolding of the zein polypeptide chain, increased hydrogen bonding and electrostatic interactions between zein and chitosan. No effects were observed on the primary structure of zein. The aggregation of complex coacervations was also reduced after treatment. With ACP treatment (40 V), resveratrol-loaded nanoparticles showed higher encapsulation efficiencies (from 51.8% to 82.7%) and better dispersion stability compared to untreated samples. A possible assembly mechanism between zein and chitosan under ACP exposure is proposed. These findings clarify the interaction between protein and polysaccharide under ACP exposure and demonstrate the promise of ACP technology in modifying zein-chitosan complex coacervation as a promising delivery carrier in the functional food field.

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.

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.

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.

Quantification of individual zein isoforms resolved by two-dimensional electrophoresis: Genetic variability in 45 maize inbred lines

A two-dimensional (2-D) electrophoresis procedure was optimized to obtain well-resolved and reproducible patterns of zein polypeptides in maize. All zein isoforms obtained through zein-specific extraction were observed from whole meal extracted with a urea/Triton/2-mercaptoethanol solution. Loading the protein samples at the acidic side of the gradient, using an amino acid solution as catholyte and running for a short time period under high Vh reduced shrinking and instability at the basic side of the isoelectric focusing (IEF) gels. Good quality and reproducible 2-D patterns were thus obtained, allowing automatic spot quantification. A linear relationship between spot intensity and zein isoform amount was established for 20 of 22 zeins detected in a 5.5-8.5 pH range using colloidal Coomassie blue staining in one maize line. The analysis of 45 genetically diverse inbred lines allowed the detection of 59 isoforms belonging to the four classes of zeins, and revealed a large qualitative and quantitative variability of individual isoforms. The classical decrease in zein amount in o2 mutant genotype was observed, and could be quantified for every isoform. The improved technique will be useful to dissect the genetic control of zein expression in maize.

Quantification of individual zein isoforms resolved by two-dimensional electrophoresis: genetic variability in 45 maize inbred lines.

A two-dimensional (2-D) electrophoresis procedure was optimized to obtain well-resolved and reproducible patterns of zein polypeptides in maize. All zein isoforms obtained through zein-specific extraction were observed from whole meal extracted with a urea/Triton/2-mercaptoethanol solution. Loading the protein samples at the acidic side of the gradient, using an amino acid solution as catholyte and running for a short time period under high Vh reduced shrinking and instability at the basic side of the isoelectric focusing (IEF) gels. Good quality and reproducible 2-D patterns were thus obtained, allowing automatic spot quantification. A linear relationship between spot intensity and zein isoform amount was established for 20 of 22 zeins detected in a 5.5-8.5 pH range using colloidal Coomassie blue staining in one maize line. The analysis of 45 genetically diverse inbred lines allowed the detection of 59 isoforms belonging to the four classes of zeins, and revealed a large qualitative and quantitative variability of individual isoforms. The classical decrease in zein amount in o2 mutant genotype was observed, and could be quantified for every isoform. The improved technique will be useful to dissect the genetic control of zein expression in maize.

Inheritance of zeins: The catalogue of zein alleles of three multigenic loci and its potential for maize breeding

Inheritance of zein components in maize has been studied by a highresolution system of acidic continuous polyacrylamide gel electrophoresis. This method revealed extraordinarily high polymorphism of zeins. Previously, three multigenic loci, Zep1, Zep2 and Zep3, encoding most zein polypeptides were identified. Four other loci control the synthesis of some minor components. Single F2 grains from 16 hybrid combinations involving 19 inbred lines were analysed. Correspondingly, 12 and 14 alleles of the Zep1 and the Zep2 loci were identified for 19 inbreds. Six alleles of the Zep3 locus were identified in five crosses. All 32 alleles of three multigenic loci were included in the catalogue of zein alleles. Genetic nomenclature for the alleles designation is proposed. Such nomenclature is in good agreement with ‘a standard for maize genetics nomenclature. The catalogue may serve for practical use and as basis for more detailed genetic studies of zeins. Practical application of the catalogue in maize breeding is discussed.

Expression of maize γ-zein and β-zein genes in transgenic Nicotiana tabacum and Lotus corniculatus

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.

Lysine-rich modified gamma-zeins accumulate in protein bodies of transiently transformed maize endosperms.

During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.

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.

Evidence against a potential endoplasmic reticulum transmembrane domain of 27K zein expressed in Xenopus oocytes

During the cellular protein targeting process, zeins (maize storage proteins) are retained in the endoplasmic reticulum (ER) where they accumulate into protein bodies. There are circumstantial and preliminary data indicating that the 27K zein, a class of zein proteins, may span the ER membrane. This potential transmembrane domain is considered very significant with regard to the mechanism of ER retention for zeins. The potential transmembrane domain may permit the 27K zein to remain in the ER and to serve as an anchor for other classes of zein, thus acting as a nucleating factor for protein body formation. This study investigated the potential transmembrane feature in a heterologous system (Xenopus laevis oocyte). Following injection of synthetic 27K zein mRNA, isolated protein vesicles were subjected to proteinase K digestion, surface biotinylation and alkaline extraction. Throughout three categories of assay the possible role of the 27K zein as a transmembrane protein was consistently refuted in this study. The 27K zein polypeptide was affected by neither proteinase K digestion nor biotinylation and was released from alkali-stripped membranes. This study, therefore, concludes that the 27K zein is not a protein body nucleating factor by virtue of an ER transmembrane feature.

Molecular analysis of wild-type and mutant alleles at the Opaque-2 regulatory locus of maize reveals different mutations and types of O2 products.

The expression of the various members of the zein multigene family in maize endosperm is controlled by different regulatory loci. One of these loci, Opaque-2, coding for a bZIP transcriptional factor, controls the expression of a subset of zein genes. Analysis of genomic DNA from plants carrying wild-type (O2) or mutant o2 alleles shows specific DNA restriction patterns that correlate with transcript types and their various gene products. Northern and western analyses show the presence in different wild types of a 1.7 kb transcript coding for different sizes of normal O2 proteins that migrate as doublets in the 68-72 kDa range. Among the various o2 mutants analysed we showed the occurrence of various null-transcript alleles, the presence of alleles with a normal size transcript which, however, produce a different-sized o2 protein, and a mutant producing both a normal size transcript and a longer transcript, but generating only a single o2 product migrating around 40 kDa. Analysis of other mutations (o7, fl2) known to affect zein polypeptide synthesis shows no interference of these mutations in the expression of the O2 gene products. The overall results indicate the occurrence of micro heterogeneity in the O2 wild-type genes and a broad spectrum of o2 mutations, both producing different sizes of O2 or o2 proteins. A nomenclature of the O2 and o2 genes based on the RFLP, transcripts and products of the various alleles is presented.

Cloning of a cDNA encoding a putative cysteine protease from germinating maize seeds

We constructed a cDNA expression library with mRNAs from the endosperm of germinating maize ( Zea mays L.) seeds to isolate sequences encoding proteases that degrade zein polypeptides. No immuno-positive plaques were detected when the library was screened with polyclonal antibodies reacting with proteases A 1–A 4, a group of zein degrading cysteine proteases isolated from germinating maize seeds. The library was subsequently screened with a cDNA (pHVEP-4) encoding the cysteine endopeptidase (EP-B) from the aleurone of germinating barley seeds. Five clones were isolated, and the longest cDNA (pMC10A) showing homology to the barley gene was characterized. This cDNA encodes a putative cysteine protease of 25 kDa, with an incomplete prosequence of 62 amino acids. There is around 70% similarity between the primary sequences of pMCP10A and pHVEP-4, with approximately 68% identity between the amino acid sequences deduced from both clones. Genes corresponding to pMCP10A are represented no more than five times in the maize genome. Transcripts hybridizing to pMCP10A are absent in the dry seed, but are present between 2 and 6 days after germination.

Role of Auxin in Maize Endosperm Development (Timing of Nuclear DNA Endoreduplication, Zein Expression, and Cytokinin).

The timing of developmental events and regulatory roles of auxin were examined in maize (Zea mays L.) endosperms. Zeatin, zeatin riboside, and indole-3-acetic acid (IAA) levels were determined by enzyme-linked immunosorbent (ELISA). Zeatin and zeatin riboside increased to maximal concentrations at an early stage (9 d after pollination [DAP]), corresponding to the stage when cell division rate was maximal. In contrast, IAA concentration was low at 9 DAP and abruptly increased from 9 to 11 DAP, thus creating a sharp decline in the cytokinin to auxin ratio. Coincident with the increase in IAA was an increase in DNA content per nucleus, attributed to postmitotic DNA replication via endoreduplication. Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D) at 5 or 7 DAP hastened the time course of DNA accumulation per nucleus and increased the average nuclear diameter, whereas 2-(para-chlorophenoxy)isobutyric acid delayed such development. Exogenously applied 2,4-D hastened the accumulation of the zein polypeptides of apparent molecular masses of 12, 14, and 16 kD and the expression of mRNA hybridizing with a zein DNA probe. We conclude that an abrupt increase in auxin induces cellular differentiation events in endosperm, including endoredupliction and expression of particular zein storage proteins.

The accumulation of zein polypeptides and zein mRNA in cultured endosperms of maize is modulated by nitrogen supply

SummaryThe effect of nitrogen (N) nutrition upon the accumulation of seed storage protein has been studied in wild‐type and opaque‐2 mutant (o2) maize endosperms grown in vitro, for 5 days, on a solid medium containing different amounts of N in the form of inorganic or organic compounds. As reference, the accumulation of zein proteins in endosperms grown to maturity in vivo under field conditions was also examined. The developmental stage up to 14–19 days after pollination (DAP) defines, both in vivo and in vitro, a phase of growth during which the major zein classes of 20 and 22 kDa are synthesized both in wild‐type and o2 endosperms. In vitro, the corresponding transcripts of the genes coding the 20 and 22 kDa zeins, and of the zein synthesis associated gene b‐32, are also present Under in vitro conditions, the level of expression of the zein synthesizing system, as monitored by zein accumulation, is dependent on the level and form of N supplied in the medium. In vivo, at stages of development from 19 DAP to maturity, the synthesis of the 22 kDa zein class and of the b‐32 protein is under control of the O2 transcriptional activator. It is hypothesized that at early stages of endosperm development (between 9 and 14–19 DAP) a ‘metabolic control’, based on N availability, is operative, possibly based on a regulatory factor different from O2.

Analyses of genes that encode the 15-kDa zein protein of maize: identification of potential gene regulatory elements

A gene ( gZ15.4.1) encoding the 15-kDa zein polypeptide was isolated from maize cultivar A5707, and its nucleotide sequence was determined. A total of 2085 bp was sequenced, including about 300 bp of 5′-flanking DNA that includes several potential regulatory elements not available in the previously published 15-kDa zein-encoding gene ( gZ15A) sequence. Several nt differences between gZ15.4.1 and gZ15A were observed, which include two single amino acid replacements.

Proposed nomenclature for the alcohol-soluble proteins (kafirins) of Sorghum bicolor (L. Moench) based on molecular weight, solubility, and structure

SDS-PAGE and differential solubility were used to demonstrate similarities between maize zein and sorghum kafirins. Polypeptides with similar apparent molecular weights were found in the zein and sorghum karafin fractions. To identify structural similarities among the karafin polypeptides and the zein polypeptides, Western blot was carried out by using α-, β-, and γ-zein antisera. A nomenclature for sorghum karafins is proposed based on similarities in molecular weight, solubility, and structure to zeins.

Hydrophobic properties of maize zein

The proteins of the zein complex from maize endosperm have been studied with the aid of hydrophobic chromatography. Their best separation was achieved on a column with TSK gel HW-65f. By comparing the results of fractionation by hydrophobic chromatography with those of electrophoresis, it was found that electrophoresis in PAAG under denaturing conditions separates the zein protein into groups according to their surface hydrophobicity. The most hydrophobic is the high-molecular-mass group of zein polypeptides.

ZEIN DEGRADATION IN THE ENDOSPERM OF MAIZE SEEDS DURING GERMINATION

The pattern and sequence of zein degradation in the endosperm of germinating maize seeds were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and by immunostaining Western blots with a monoclonal antibody to α‐zein and polyclonal antibodies to β‐, γ‐, and δ‐zeins. The results indicated: 1) the degradation of the predominant α‐zein fraction (23.8 and 26.7 kD) started on the 5th day after germination (DAG) and continued gradually until the 10th DAG with a small amount remaining undegraded up to the 26th DAG; 2) β‐zein (17 kD) began to be degraded on the 2d DAG, and the degradation of the 17 kD polypeptide was completed by the 7th DAG; 3) γ‐zeins (27 and 18 kD) were the first zeins to be degraded, and the degradations of γ‐zeinl (27 kD) and γ‐zein2 (18 kD) were complete by the 3rd and 4th DAG, respectively; and 4) the degradation of δ‐zein (10 kD) began on the 4th DAG and was complete by the 7th DAG. Based on these results, the following arrangement of zein polypeptides within the protein bodies is postulated, assuming that the proteolytic events start at the periphery and processed towards the core of the protein body: 1) γ‐zeins are situated around the periphery of the protein bodies and are possibly a structural component of the protein body membrane or directly anchored in the membrane; 2) β‐zein would be internal to γ‐zeins; and 3) α‐zein and δ‐zein would be in the protein body core. This arrangement is largely consistent with published data on the immunocytochemical localization of zeins, and it indicates that the different classes of zein are not randomly organized within the matrix of the protein body.

Linkages among zein genes determined by isoelectric focusing

Genetic control of the major zein polypeptides in maize (Zea mays L.) was studied by isoelectric focusing (IEF) in agarose. Linkage relationships were determined by making a number of crosses, then determining the expression of zein polypeptides in backcross seeds. Chromosome linkages were determined by using the markers sugary-1 (for chromosome 4), yellow-8, and a waxy 7-9 translocation (for chromosome 7). Nine zeins were in one linkage group on chromosome 4, six in another linkage group on chromosome 4, and four zeins were in one linkage group on chromosome 7. Some IEF single bands consisted of at least two polypeptides, which were detected by subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis, by aberrant ratios in backcrosses, or by differing recombination percentages. One zein occurred only in homozygous sugary-1 seeds. Three sets of closely-linked zeins were noted that occurred together almost exclusively in certain inbreds.