Structure of maize protein bodies and immunocytochemical localization of zeins

Summary Zeins, the seed storage proteins of maize (Zea mays L.), are synthesized by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum in developing endosperm, where they assemble into protein bodies. To better understand the organization of protein bodies and the mechanism by which zeins are assembled, we have used immunolocalization to study their distribution within isolated protein bodies. In sections stained with uranyl acetate and lead citrate, the protein body matrix consists of light- and dark-staining regions with the darker stain predominating at the periphery and the lighter stain in the central region. Immunogold staining of the storage proteins in isolated protein bodies reveals a distinct segregation with -zein localized in the light-staining region and - and -zein localized in the dark-staining regions. However, the relative amounts and distribution of these proteins varies substantially among different protein bodies. These results indicate a more complex internal organization than has been previously observed, and suggest that spatial and/or temporal differences in zein synthesis account for this complexity.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - PB phosphate buffer - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TTBS Tween-20/tris-buffered saline - TBS-T Tris-buffered saline/Tween-20 - TBS-T-B Tris-buffered saline/Tween-20/bovine serum albumin     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering     

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.

     

Evidence for the presence of two different types of protein bodies in wheat endosperm

Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.     

A novel vesicle derived directly from endoplasmic reticulum is involved in the transport of vacuolar storage proteins in rice endosperm

We found novel vesicles derived from rough endoplasmic reticulum (ER) in rice endosperm. The novel vesicles had characteristic structures different from that of the ER-derived protein body type I and the Golgi-derived dense vesicles. Immunocytochemical analysis revealed that the novel vesicles are derived directly from the aggregates of vacuolar storage proteins in the rough ER. In addition, BiP, an ER-resident molecular chaperone, was localized in the novel vesicles, but also in protein storage vacuoles (PSVs). These results suggest that the novel vesicles mediate transport of vacuolar storage proteins directly from the ER to PSVs in rice endosperm.     

Protein accumulation in developing endosperm of a high-protein line of Triticum dicoccoides

Abstract. Endosperm tissue from developing grains of a line of wheat ( Triticum dicoccoides ) which accumulates up to 30% protein in the mature grain, was examined by electron microscopy to establish the ontogeny of the storage protein bodies. Ultrastructural evidence suggests that storage proteins of wheat may be transported from their site of synthesis on the rough endoplasmic reticulum (ER) to protein bodies by two different routes within the endomembrane system. The first route, which probably functions throughout protein deposition, involves the transport of protein from the cisternal rough ER to the protein vacuoles via the Golgi apparatus. The second route, observed 20 d after anthesis, appears to lead directly from dilated regions of the rough ER to protein vacuoles, bypassing the dictyosomes. Phytin inclusions are found in protein vacuoles of starchy endosperm cells adjacent to the aleurone layer of developing grain.     

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.     

Protein Bodies at Early Stages of Development in High Lysine Mutant Notch-2 and Parent NP-113 Barley

In barley parent NP-113, endospermic protein bodies originate on rough endoplasmic reticulum, either as electron transluscent vesicles or as very small, spherical, electron dense protein bodies, These are translocated to vacuoles tor enlargement and subsequent storage, Endospermic protein bodies of Notch-2 high lysine mutant are either vacuolar, or confined to distended cisternae of smooth endoplasmic reticulum. Vacuolar protein bodies are of two types one, flocculent, which loosely fill up almost the entire vacuolar space; two, spherical, relatively compact and granular, Protein bodies, confined to smooth endoplasmic reticulum are small, spherical, electron dense or electron transluscent, These protein bodies fuse to form electron dense proteinaceous masses which are deposited in the cytosol due to disruption of the confining smooth endoplasmic reticulum.     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum

The ultrastructure of protein deposition in the starchy endosperm of developing rice (Oryza sativa L.) grains was examined in conventionally fixed (glutaraldehyde and osmium tetroxide) tissues and also in thick sections (0.3 m) of zinc iodide-osmium tetroxide post-fixed tissue. Three types of previously characterised protein body were observed and it was shown that each type was initiated by dilations of the endoplasmic reticulum. Crystalline type protein bodies were initiated by a ribosome-free dilation from rough cisternal endoplasmic reticulum and developed by inclusion of protein from dictyosome-derived vesicles. The large spherical and small spherical protein bodies developed within the cisternae of the rough endoplasmic reticulum.Abbreviations Cr crystalline protein body - DAF days after fertilization - ER endoplasmic reticulum - Ls large spherical protein body - Ss small spherical protein body - ZIO zinc iodide-osmium tetroxide     

Effect of thefloury-2 locus on protein body formation during maize endosperm development

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. These proteins, collectively called zeins, are translocated into the lumen of the rough endoplasmic reticulum, where they assemble into protein bodies. Protein body formation in normal genotypes occurs via an ordered deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about 1 m. These structures consist of a central core that contains predominantly -zein; this central region is surrounded by a peripheral layer of - and -zeins, and the entire structure is bounded by rough endoplasmic reticulum.In the endosperm mutant floury-2 the levels of all classes of zeins are reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype observed in normal genotypes. In contrast to the discrete, spherical protein bodies which are formed in normal maize endosperm, the protein bodies within floury-2 endosperm are irregular and the zeins are disorganized; patches of - and -zeins occur within irregularly lobed clusters of -zein within the lumen of the rough endoplasmic reticulum. The implications of this aberrant distribution are discussed, both with respect to protein body development and kernel characteristics.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid     

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

Summary 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 m. 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 m 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.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

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.     

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.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

Summary A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.Abbreviations BiP binding protein - BSA bovine serum albumin - De*-B 30 Defective endosperm B 30 - DAP day after pollination - ER endoplasmic reticulum - fl 2 floury-2 - hsp 70 70 kDa heat shock protein - Mc Mucronate - TBST 20mM Tris-HCl, pH8.2 at 20°C, 500mM NaCl, 0.3% Tween 20 - TBST-B TBST with 1% (w/v) BSA     

Unexpected deposition patterns of recombinant proteins in post-endoplasmic reticulum compartments of wheat endosperm

Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals.     

The accumulation of alpha-zein in transgenic tobacco endosperm is stabilized by co-expression of beta-zein

The cysteine-poor alpha-zein is the major prolamin storage protein fraction in maize endosperm and is localized in the interior of protein bodies with delta-zein, whereas the hydrophobic cysteine-rich beta- and gamma-zein are found on the exterior of the PB. In transgenic tobacco endosperm expressing zein genes, alpha-zein was unstable unless co-expressed with gamma-zein. Here we showed that alpha-zein was also stabilized by beta-zein. Small accretions of alpha- and beta-zeins, similar in appearance to maize protein bodies, were localized to the endoplasmic reticulum within tobacco endosperm cells. The zein proteins were also localized to protein storage vacuoles in a more dispersed pattern, suggesting that they were transported there after they were post-translationally sequestered into the ER.