Formation of protein storage bodies during embryogenesis in cotyledons of Sinapis alba L.

An electron microscopic investigation of fine structural changes in post-meristematic cotyledon mesophyll cells during the period of storage protein accumulation (16–32 d after pollination) showed that the rough ER, the Golgi apparatus and the developing vacuome are intimately involved in the formation of storage protein bodies (aleurone bodies). At the onset of storage protein accumulation (16–18 d after pollination) storage protein-like material appears within Golgi vesicles and preformed vacuoles. At a later stage (24 d after pollination) similar material can also be detected within vesicles formed directly by the rough endoplasmic reticulum (ER). It is concluded that there are two routes for storage protein transport from its site of synthesis at the ER to its site of accumulation in the vacuome. The first route involves the participation of dictyosomes while the second route bypasses the Golgi apparatus. It appears that the normal pathways of membrane flow in the development of central vacuoles in post-meristematic cells are used to deposit the storage protein within the protein bodies. Thus, the protein body can be regarded as a transient stage in the process of vacuome development of these storage cells.Abbreviation ER endoplasmic reticulum     

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.     

Gluten quality of bread wheat is associated with activity of RabD GTPases

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER‐to‐Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down‐regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT‐PCR to be down‐regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE‐HPLC found that a smaller proportion of HMW‐GS and large proportion of LMW‐GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS‐PAGE was seen in the transgenic grain.     

Wheat (Triticum aestivum L.) [gamma]-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat [gamma]-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the [gamma]-gliadin was transported to the yeast vacuoles. When a deletion mutant of the [gamma]-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the [gamma]-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.     

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.     

The Ultrastructural Evidence on the Origin of Protein Bodies in the Rough Endoplasmic Reticulum of Developing Cotyledons of Soybean

The process of protein body formation from rough endoplasmicreticulum (RER) in cotyledon cells of soybean has been followed.From about 43 d after flowering (DAF), the lumen of some partsof RER cisternae was dilated and filled gradually with proteinaceousmaterial. This kind of dilated RER expanded further to formlong and irregularly shaped protein bodies (PBs), and the latterdivided into smaller spherical protein bodies in mature seedsat 63 DAF. From these results and our previous observations,we draw the conclusion that there are two pathways of proteinbody formation in developing cotyledon cells in soybean. Duringthe early stage, vacuoles in the cells were filled with proteinaceousmaterial and turned into protein bodies. During the late stage,some of the dilated RER with storage proteins developed intoPBs A few vacuoles can also form PBs at this late stage. Inaddition, some fibre structures, 7–8 nm in width, wereseen to be oriented in parallel in longitudinal sections ofRER cisternae in cotyledon cells at 45 DAF. Soybean, protein body, ER origin, storage protein     

Evidence for a novel route of wheat storage proteins to vacuoles

Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.     

Assembly and transport of seed storage proteins

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.     

Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons

Development of legume seeds is accompanied by the synthesis of storage proteins and lectins, and the deposition of these proteins in protein-storage vacuoles (protein bodies). We examined the subcellular distribution, in developing seeds of the common bean, Phaseolus vulgaris L., of the major storage protein (phaseolin) and the major lectin (phytohemagglutinin, PHA). The proteins were localized using an indirect immunocytochemical method in which ultrathin frozen sections were immunolabeled with rabbit antibodies specific for either PHA or phaseolin. Bound antibodies were then localized using goat-anti-rabbit immunoglobulin G adsorbed onto 4- to 5-nm colloidal gold particles. The sections were post-fixed with OsO₄, dehydrated, and embedded in plastic on the grids. Both PHA and phaseolin exhibited a similar distribution in the storage-parenchyma cells, being found primarily in the developing protein bodies. Endoplasmic reticulum and Golgi complexes (cisternal stacks and associated vesicles) also were specifically labeled for both proteins, whereas the cytosol and other organelles, such as mitochondria, were not. We interpret these observations as supporting the hypothesis that the transport of storage proteins and lectins from their site of synthesis, the rough endoplasmic reticulum, to their site of deposition, the protein bodies, is mediated by the Golgi complex.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - PBS phosphate-buffered saline - PHA phytohemagglutinin     

Origin and development of protein bodies in cotyledons of Vicia faba

Storage proteins of the field bean (Vicia faba L., var. minor, cv. “Fribo”) are synthesized and accumulated in the cotyledons during stage 2 of seed development. Deposition of protein reserves takes place in the protein bodies. The generation of protein bodies was investigated electronmicroscopically using ultra-thin sections as well as the freeze-fracturing technique. During the initial period of storage protein formation, globulins are deposited in large vacuoles which later are transformed to give increasing numbers of small vacuoles with decreasing size. The vacuoles disappear early during the stage of storage protein formation and generate the first protein bodies. During the subsequent period of maximum storage protein formation, which takes place at the rough endoplasmic reticulum (rER), swollen ER strands appear which seem to be entirely filled with protein, and these generate ER-produced protein vacuoles (ERPVAC). The vesicles are transformed in a manner comparable to the vacuoles in the initial period of developmental stage 2 and thus generate the major quantity of protein bodies. Both processes seem to represent only two variants of an uniform mechanism of protein body generation.     

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.     

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.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains.

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.     

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     

Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.Abbreviations Con A concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G     

The N- and C-terminal regions regulate the transport of wheat gamma-gliadin through the endoplasmic reticulum in Xenopus oocytes.

Following sequestration into the endoplasmic reticulum (ER), wheat storage proteins are naturally either retained and packaged into protein bodies within this organelle or exported to the Golgi apparatus. To identify protein domains that control the sorting of wheat storage proteins within the ER, a wild-type gamma-gliadin storage protein as well as two of its deletion mutants, each bearing either of the two autonomous N- and C-terminal regions, were expressed in Xenopus oocytes. Our results demonstrated that the N-terminal region of the gliadin, which is composed of several tandem repeats of the consensus sequence PQQPFPQ, was entirely retained within the ER and accumulated in dense protein bodies. In contrast, the C-terminal autonomous region was efficiently secreted to the medium. The wild-type gamma-gliadin, containing both regions, was secreted at a lower rate and less efficiently than its C-terminal region. These results suggest that sorting of the wheat gamma-gliadin within the ER may be determined by a balance between two opposing signals: one functions in the retention and packaging of the storage protein within the ER, while the second renders the protein competent for export from this organelle to the Golgi apparatus.     

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.     

Multivesicular bodies in developing tobacco seed and mung bean are functionally equivalent

Protein storage vacuoles (PSVs) are the primarily storage organelles in cotyledon cells for protein preservation in seeds. Storage proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus for subsequent delivery to PSVs via presumably Golgi-derived dense vesicles (DVs). However, recent studies demonstrated that storage proteins in early stage of developing cotyledon of mung beans reached the multivesicular bodies (MVBs) prior to the detection of DVs, indicating the possible involvement of MVBs in mediating transport of storage proteins during the early stage of seed development. Here, we further show that the MVBs in developing tobacco seeds are functionally and biochemically equivalent to those in developing mung beans. Thus, MVBs in developing tobacco seeds are structurally distinct from DVs, contain both vacuolar sorting receptors (VSRs) and storage proteins, and they are insensitive to treatments of wortmannin and brefeldin A (BFA).     

BiP, HSP70, NDK and PDI in wheat endosperm. I. Accumulation of mRNA and protein during grain development

Biosynthesis and accumulation of seed storage proteins such as the wheat glutens depend on the activity of a variety of other proteins, including chaperones and foldases. cDNA probes and antibodies to two chaperone proteins and a foldase were used to follow mRNA and protein accumulation in developing grains of wheat ( Triticum aestivum , cvs Cheyenne and Butte). Endosperm was separated from other grain components and protein accumulation was analyzed on a per mg fresh weight basis. The ER resident chaperone BiP (binding protein) and foldase PDI (protein disulfide isomerase) accumulated to maximal levels in the middle stage of endosperm development, a period of rapid cell expansion and storage protein accumulation, whereas levels of a cytosolic chaperone, HSP70, remained relatively constant throughout grain development. In contrast, nucleoside diphosphate kinase (NDK), a cytosolic enzyme needed for synthesis of nucleoside triphosphates, accumulated early in endosperm development during the period of nuclear division and cell formation. When analyzed as a fraction of total protein the relative abundance of all four proteins peaked early in grain development and then declined. Accumulation of mRNA for the four proteins also peaked early in grain development. Although BiP and PDI formed a declining percentage of total protein as storage protein accumulated, their pattern of accumulation was compatible with a proposed role as catalysts for storage protein folding and accumulation in the ER.