Zein Synthesis Research Articles

Allelic Diversity and Development of Breeder-Friendly Marker Specific to floury2 Gene Regulating the Accumulation of α-Zeins and Essential Amino Acids in Maize Kernel.

Maize zeins lack essential amino acids, such as methionine, lysine, and tryptophan. The floury2 (fl2) mutation reduces zein synthesis and increases methionine and lysine content in kernels. In this study, fl2 gene (1612bp) was sequenced in eight wild-type and two mutant inbreds and detected 218 SNPs and 18 InDels. Transversion of C to T at 343bp position caused the substitution of alanine by valine in the fl2 mutant. A PCR-based marker (FL-SNP-CT) was developed, which distinguished the favorable mutant fl2 allele (T) from the wild-type (C) Fl2 allele. Gene-based diversity analysis using seven gene-based InDel markers grouped 48 inbred lines into three major clusters, with an average genetic dissimilarity coefficient of 0.534. The average major allele frequency, gene diversity, heterozygosity, and polymorphism information content of the InDel markers were 0.701, 0.392, 0.039, and 0.318, respectively. Haplotype analysis revealed 29 haplotypes of fl2 gene among these 48 inbreds. Amino acid substitution (Ala-Val) at the signal peptide cleavage site produced unprocessed 24-kDa mutant protein instead of 22-kDa zein found in normal genotype. Eight paralogues of fl2 detected in the study showed variation in exon lengths (616-1170bp) and translation lengths (135-267 amino acids). Orthologue analysis among 15 accessions of Sorghum bicolor and two accessions of Saccharum spontaneum revealed a single exon in fl2 gene, ranging from 267 to 810bp. The study elucidated the molecular basis of fl2 mutation and reported a breeder-friendly marker for molecular breeding programs. This is the first study to characterize fl2 gene in a set of subtropically adapted inbreds.

Enzymatic synthesis of self-adhesive zein to impart durable hydrophobicity with antioxidant activity to cotton fabrics

In this study, dopamine (DA) was used as the acyl receptor to modify zein through transglutaminase (TGase)-mediated reaction, producing self-adhesive zein (zein-DA complex). The structure of zein-DA complex was characterized by Fourier transform infrared spectrum (FT-IR), 1H nuclear magnetic resonance spectrum (1H NMR), and circular dichroism spectrum (CD), revealing the successful grafting of catechol structure of DA to zein. The results of SDS-PAGE demonstrated that zein-DA complex exhibited great ability of self-polymerization. The zein-DA complex absorbed on the cotton fabric surface imparted hydrophobicity with a water contact angle value (WCA) of 121.34 ± 1.14° to the fabrics. After 5 washing cycles, the WCA still remained 100.28 ± 4.83°, which indicated the fabrics obtained relatively durable hydrophobicity due to the self-adhesion of zein-DA complex. The results of FT-IR, scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and color reaction tests manifested that much more protein attached on the surface of cotton fabric treated with zein-DA complex after washing compared with that with zein, which verified that the zein-DA complex had strong adhesion properties. Due to the introduction of DA, the relative DPPH free radical scavenging activity of cotton treated with zein-DA complex reached 43.60 ± 0.28 %, which was obviously higher than cotton treated with zein. The functionalized fabrics with zein-DA complex had both hydrophobicity and antioxidant activities.

Mechanisms underlying the formation of non-thermal deamidated zein via synergistic treatment of cold plasma with acid: Improved foam performance

Zein with a modified fibrosis conformation has been demonstrated to be an effective surface component for stabilizing Pickering foam. In this study, a non-thermal deamidation method was developed using cold plasma (CP) in synergy with hydrochloric acid (HCl), tartaric acid (TA), and gluconolactone (GDL). The structure and properties of the material were investigated to better understand the mechanism of non-thermal deamidated zein synthesis. The results demonstrated that within 12 min, the degrees of deamidation (16.21, 14.14, and 8.35%) were regulated by the varying proton catalytic rates of HCl, TA, and GDL in the CP field. Following treatment, notable enhancements were observed in the solubility (0.12–0.28 mg/mL), foamability (50.00–166.67%), and foaming stability (106.67–128.00%) (P < 0.05). The increase in β-sheet, random coil, and loose structure of deamidated zein was revealed by employing FTIR and UV spectroscopy. The unfolding of structure resulted in the exposure of hydrophobic groups, thereby enhancing the protein's foaming properties. Deamidated zein has increased interfacial adsorbability, which was demonstrated by atomic force microscope and scanning electron microscopy. Better interface performance was shown by CP synergistic TA treatment for 12 min (CPT12-zein), offering the possibility of commercial manufacturing of foamed foods in the future.

The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize.

Endosperm filling in maize (Zea mays), which involves nutrient uptake and biosynthesis of storage reserves, largely determines grain yield and quality. However, much remains unclear about the synchronization of these processes. Here, we comprehensively investigated the functions of duplicate NAM, ATAF1/2, and CUC2 (NAC)-type transcription factors, namely, ZmNAC128 and ZmNAC130, in endosperm filling. The gene-edited double mutant zmnac128 zmnac130 exhibits a poorly filled kernel phenotype such that the kernels have an inner cavity. RNA sequencing and protein abundance analysis revealed that the expression of many genes involved in the biosynthesis of zein and starch is reduced in the filling endosperm of zmnac128 zmnac130. Further, DNA affinity purification and sequencing combined with chromatin-immunoprecipitation quantitative PCR and promoter transactivation assays demonstrated that ZmNAC128 and ZmNAC130 are direct regulators of 3 (16-, 27-, and 50-kD) γ-zein genes and 6 important starch metabolism genes (Brittle2 [Bt2], pullulanase-type starch debranching enzyme [Zpu1], granule-bound starch synthase 1 [GBSS1], starch synthase 1 [SS1], starch synthase IIa [SSIIa], and sucrose synthase 1 [Sus1]). ZmNAC128 and ZmNAC130 recognize an additional cis-element in the Opaque2 (O2) promoter to regulate its expression. The triple mutant zmnac128 zmnac130 o2 exhibits extremely poor endosperm filling, which results in more than 70% of kernel weight loss. ZmNAC128 and ZmNAC130 regulate the expression of the transporter genes sugars that will eventually be exported transporter 4c (ZmSWEET4c), sucrose and glucose carrier 1 (ZmSUGCAR1), and yellow stripe-like2 (ZmYSL2) and in turn facilitate nutrient uptake, while O2 plays a supporting role. In conclusion, ZmNAC128 and ZmNAC130 cooperate with O2 to facilitate endosperm filling, which involves nutrient uptake in the basal endosperm transfer layer (BETL) and the synthesis of zeins and starch in the starchy endosperm (SE).

Quality protein maize: nutritional and bioactive composition, technological attributes and potential food applications

SummaryThe present review aimed to explain the composition, nutritional importance, technological functionality, morphology, rheological and bioactive profile of quality protein maize (QPM). This review further highlights the potentiality of QPM for the development of versatile value‐added products, which are advantageous for consumers, researchers and industries related to this area. Literature searches were conducted using PubMed, Research gate and Google scholar. Full‐text screening of 112 articles was carried out, resulting in a final sample of 90 articles of which the most reliable and recent articles were thoroughly reviewed. Quality protein maize contains the opaque‐2 gene that limits the zein synthesis meanwhile promoting the synthesis of other non‐zein proteins that are rich in tryptophan and lysine. QPM has high antioxidative properties that prevent cardiovascular and degenerative disorders. Regular intake of QPM can also reduce the symptoms of protein–energy malnutrition. Thus, QPM has an excellent potential to be utilised as an ingredient or enrichment source in several food categories.

3D Electron Microscopy Gives a Clue: Maize Zein Bodies Bud From Central Areas of ER Sheets.

Zeins are the main storage proteins in maize seed endosperm, and the onset of zein synthesis in young seeds challenges the endomembrane system and results in the formation of storage organelles. Even though zeins lack a conventional endoplasmic reticulum (ER) retention signal, they accumulate within the ER and assemble in conspicuous ER-derived protein bodies (PBs) stabilized by disulfide bridge formation and hydrophobic interaction between zein chains. Zein body formation during seed development has been extensively studied, as well as the mechanisms that lead to the initiation of PBs. However, the exact course of the PB formation process and the spatial relationship with the ER remain unclear. The development of serial block face scanning electron microscopy (SBF-SEM) techniques that allow three-dimensional imaging combined with the high resolution of electron microscopy provides new perspectives on the study of the plant endomembrane system. Here, we demonstrate that (i) the ER of maize seeds is mainly formed by massive sheets and (ii) PBs are not budding from tubules or the edge of sheets, but protrude from the entire surface of the ER sheet.

Responses to Hypoxia and Endoplasmic Reticulum Stress Discriminate the Development of Vitreous and Floury Endosperms of Conventional Maize (Zea mays) Inbred Lines

Major nutritional and agronomical issues relating to maize (Zea mays) grains depend on the vitreousness/hardness of its endosperm. To identify the corresponding molecular and cellular mechanisms, most studies have been conducted on opaque/floury mutants, and recently on Quality Protein Maize, a reversion of an opaque2 mutation by modifier genes. These mutant lines are far from conventional maize crops. Therefore, a dent and a flint inbred line were chosen for analysis of the transcriptome, amino acid, and sugar metabolites of developing central and peripheral endosperm that is, the forthcoming floury and vitreous regions of mature seeds, respectively. The results suggested that the formation of endosperm vitreousness is clearly associated with significant differences in the responses of the endosperm to hypoxia and endoplasmic reticulum stress. This occurs through a coordinated regulation of energy metabolism and storage protein (i.e., zein) biosynthesis during the grain-filling period. Indeed, genes involved in the glycolysis and tricarboxylic acid cycle are up-regulated in the periphery, while genes involved in alanine, sorbitol, and fermentative metabolisms are up-regulated in the endosperm center. This spatial metabolic regulation allows the production of ATP needed for the significant zein synthesis that occurs at the endosperm periphery; this finding agrees with the zein-decreasing gradient previously observed from the sub-aleurone layer to the endosperm center. The massive synthesis of proteins transiting through endoplasmic reticulum elicits the unfolded protein responses, as indicated by the splicing of bZip60 transcription factor. This splicing is relatively higher at the center of the endosperm than at its periphery. The biological responses associated with this developmental stress, which control the starch/protein balance, leading ultimately to the formation of the vitreous and floury regions of mature endosperm, are discussed.

Review: bio-based films from zein, keratin, pea, and rapeseed protein feedstocks

The development of bio-based films from proteins for their utilization as packaging materials and composites has gained popularity due to the availability, renewable nature, and biodegradability of proteins. The most studied of these polymers is sourced from soy. However, other sources of proteins show great commercial potential when transformed into films. As such, zein from corn and keratin from low-value chicken feather can be plasticized into films which exhibit good mechanical properties. Films derived from pea proteins can resist UV light transmission due to the presence of disulfide bonds within the network and the presence of amino acids such as tyrosine, tryptophan, and phenylalanine. Proteins from rapeseed have not been extensively studied as feedstocks for bio-based films but show great potential since their amino acid composition is similar to soy proteins. This review aims at combining and condensing the body of research performed on the synthesis of zein-, keratin-, pea-, canola-, and rapeseed-based films. The structural diversity of these proteins highly influences their processing methods and the physical properties of the resulting films. The potential applications of these bio-based materials are also highlighted.

Kernel lysine content does not increase in some maize opaque2 mutants

The recessive mutant allele of the opaque2 gene (o2) alters the endosperm protein pattern and increases the kernel lysine content of maize (Zea mays L.). In this study, sequencing results showed that the o2 mutant was successfully introgressed into 12 elite normal maize inbred lines by marker assisted selection (MAS). The average genetic similarity between these normal inbred lines and their o2 near-isogenic lines (NILs) was more than 95%. Kernel lysine content increased significantly in most of o2 NILs lines relative to normal elite inbreds, but remained unchanged in the genetic backgrounds Dan598o2 and Liao2345o2. Moreover, the kernel characteristics of these two o2 NILs did not differ from the other inbred lines. The results of lysine content analysis in the F1 hybrids between Liao2345o2 and Dan598o2 and other o2 NILs demonstrated that gene(s) other than opaque2 may control kernel lysine content in these two o2 NILs. The results of zein analysis showed that 22-kD α-zein synthesis was reduced or absent, and the 19-kD α-zein synthesis was greatly reduced compared with the recurrent parents in most o2 NILs except for Dan598o2 and Liao2345o2. Our results indicate that gene(s) other than opaque2 may play more important roles in zein synthesis and kernel lysine content in some maize genetic backgrounds.

Synthesis and characterisation of zein–curcumin colloidal particles

Zein–curcumin composite colloidal particles were synthesized using an antisolvent precipitation method. The average particle size could be controlled down to 100–150 nm, depending on the solvent system and the ratio of zein and curcumin used in the synthesis. In all cases, spherical particles were obtained, as confirmed by transmission electron microscopy. Depending on the preparation conditions, curcumin load and encapsulation efficiency varied from 1.6 to 4.1% and 71.1 to 86.8%, respectively. Solid state characterization by differential scanning calorimetry and X-ray diffraction indicated the amorphous nature of entrapped curcumin. An UV irradiation study confirmed enhanced photostability of curcumin due to the entrapment of curcumin in the biopolymeric matrix. The particles were also found to have good colloidal stability at a broad range of physiologically relevant pH (1.2, 4.5, 6.7 and 7.4) and in simulated gastro-intestinal conditions. Results from an in vitro mucoadhesion study showed retention of more than 60% curcumin for 150 minutes. The mucoadhesion property was further confirmed from a mucin association study carried out on Caco-2 cells.

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.

The Maize Floury1 Gene Encodes a Novel Endoplasmic Reticulum Protein Involved in Zein Protein Body Formation

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.

A defective signal peptide in a 19-kD alpha-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant.

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel alpha-zein protein in De*-B30 that migrates between the 22- and 19-kD alpha-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD alpha-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD alpha-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD alpha-zein protein.

Quantitative trait locus mapping of loci influencing elongation factor 1alpha content in maize endosperm.

The nutritional value of maize (Zea mays) seed is most limited by its protein quality because its storage proteins are devoid of the essential amino acid lysine (Lys). The Lys content of the kernel can be significantly increased by the opaque-2 mutation, which reduces zein synthesis and increases accumulation of proteins that contain Lys. Elongation factor 1alpha (eEF1A) is one of these proteins, and its concentration is highly correlated with the Lys content of the endosperm. We investigated the genetic regulation of eEF1A and the basis for its relationship with other Lys-containing proteins by analyzing the progeny of a cross between a high (Oh51Ao2) and a low (Oh545o2) eEF1A maize inbred. We identified 83 simple sequence repeat loci that are polymorphic between these inbreds; the markers are broadly distributed over the genome (1,402 cM) with an average interval of 17 cM. Genotypic analysis of the F(2) progeny revealed two significant quantitative trait loci that account for 25% of the variance for eEF1A content. One of these is on the short arm of chromosome 4 and is linked with a cluster of 22-kD alpha-zein coding sequences; the other quantitative trait locus is on the long arm of chromosome 7. The content of alpha-zein and gamma-zein was measured in pools of high- and low-eEF1A individuals obtained from this cross, and a higher level of alpha-zein was found to cosegregate with high eEF1A content. Allelic variation at the 22-kD alpha-zein locus may contribute to the difference of eEF1A content between Oh51Ao2 and Oh545o2 by increasing the surface area of protein bodies in the endosperm and creating a more extensive network of cytoskeletal proteins.

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.

Nitrogen and hormonal responsiveness of the 22 kDa alpha-zein and b-32 genes in maize endosperm is displayed in the absence of the transcriptional regulator Opaque-2.

The maize (Zea mays L.) b-ZIP transcriptional activator Opaque-2(O2) regulates the synthesis of major endosperm proteins. In the o2 homozygote, 22 kDa zein prolamins and the b-32 ribosome-inactivating protein are greatly reduced in level. An in vitro endosperm culture system has been studied in which o2 endosperm synthesizes 22 kDa zein and b-32 in response to nitrogen supplements. An increase in 22 kDa zein mRNA concentration is also seen, implying an effect at the level of transcription or differential RNA turnover. The nitrogen-dependent induction of 22 kDa zein synthesis in cultured o2 endosperm was further investigated by analysing transient expression of reporter constructs. The highest response to nitrogen was exhibited by the intact 22 kDa zein promoter. Removal of individual O2 binding sites either reduced or increased overall promoter activity, but always decreased the nitrogen-dependent stimulation of activity. This effect was observed equally in wild-type and o2 mutant endosperm. It is concluded that a factor other then O2 is responsible for activating the 22 kDa zein promoter under high-nitrogen culture conditions. Despite its occurrence in the absence of O2 protein, the nitrogen response is mediated through binding at O2 binding sites. An induction of 22 kDa zein and b-32 synthesis in cultured o2 endosperm could also be achieved on nitrogen-free media by the addition of abscisic acid or methyl jasmonate.

Genetic manipulations of protein quality in maize grain

This paper considers the main aspects of the genetic organization of the synthesis of zeins in maize endosperm. Recent advances in zein chemistry and biochemistry are discussed and the organization of zein structural genes is presented with particular emphasis on their arrangement within a multigene family. In addition, we present progress toward understanding the regulation of zein gene expression in relation to opaque loci. Finally, we focus our attention to genetic and molecular approaches for upgrading grain protein quantity and quality of maize grains.

Three-Dimensional reconstruction of protein bodies in developing maize endosperm using immunocytochemical techniques

The storage proteins of maize (Zea mays L.), like many other cereals, are a group of alcohol-soluble proteins classified as prolamines. In maize these proteins comprise four structurally distinct types and are termed zeins. Zein synthesis is initiated in developing maize endosperm and continues until the seed reaches maturity (between approximately 12 to 50 days after pollination). Zeins are synthesized by polysomes bound to the rough endoplasmic reticulum (RER) and they accumulate as insoluble aggregates called protein bodies. Our previous studies have shown that the zeins are deposited within protein bodies in a defined order in normal genotypes. However, the actual three-dimensional organization of the zeins within protein bodies and the interactions that occur between the various zeins is not known. Our goal is to understand protein body organization and the interactions that occur between the various proteins.The four types of zeins are identified by their molecular weight after separation by SDS-PAGE, and are designated α-, β-, γ- and 6-zeins. The β-, γ-, and δ-zeins are all sulfur-rich proteins, while the α-zeins contain only low amounts of cysteine and methionine. These proteins can be individually detected by immunocytochemical staining of ultrathin sections. Additionally, ultrathin sections poststained with lead citrate and uranyl acetate demonstrate that there are two distinct regions that correlate with the immunocytochemical staining patterns.

The origin of lysine-containing proteins in opaque-2 maize endosperm

The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 micrograms of lysine compared with 350 micrograms in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

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.