The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.     

Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.     

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.     

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.     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.     

Zeolin. A new recombinant storage protein constructed using maize gamma-zein and bean phaseolin

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of gamma-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type gamma-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the gamma-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.     

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.     

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.     

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.     

Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.     

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.     

The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 alpha-zein

Zeins are the major storage proteins in corn seeds organized in protein bodies located in the endosperm. They are soluble in alcoholic solution and depict a high tendency to aggregation. The Z19 alpha-zein aggregates obtained by heating show a particular and interesting temperature-dependent behavior. This work was aimed at determining not only the effect of temperature on the aggregation behavior, but also the effect of the sulfhydryl groups and disulphide bonds on the thermal aggregation process under non-aqueous conditions. Z19 alpha-zein was chemically modified to obtain different sulfhydryl groups and disulphide-bonds content. Far-UV CD, ANS emission fluorescence, and dynamic light scattering, as well as differential scanning calorimetry, were performed to characterize this protein. Removal of these disulphide-bonds and alkylation of all the sulfhydryl groups in the protein promoted the lowest T(m) of 57.36 degrees C, eliminated aggregation, enhanced protein flexibility, and diminished thermal stability. These results suggest that the disulphide linkage could be the driving force for the Z19 alpha-zein aggregation.     

Expression of zein in long term endosperm cultures of maize

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.     

Polypeptide compositions and NH2-terminal amino acid sequences of proteins in foxtail and proso millets

Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH2-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH2-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to alpha-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of gamma-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.