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     

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     

Early Stages in Wheat Endosperm Formation and Protein Body Initiation

The early stages of endosperm formation and protein body initiationare described for hard red winter wheat using light and transmissionelectron microscopy. Two days after flowering (DAF) the endospermwas a thin layer of coenocytic cytoplasm lining the embryo sac.By 4 DAF the endosperm had cellularized and completely filledthe embryo sac. Enough differentiation had occurred by 6 DAFto distinguish cells destined to become the aleurone layer,sub-aleurone region and central endosperm. Protein bodies wereinitiated at about 6–7 DAF and were first found near theGolgi apparatus. Wheat was ready for combine harvest at 34 DAF.Enlargement of the small protein bodies near the Golgi apparatusoccurred by several mechanisms: (1) fusion with one or moreof the dense Golgi vesicles or fusion with other protein bodies,(2) fusion with small electron-lucent Golgi-derived vesicles,(3) pinocytosis of a portion of the adjacent cytoplasm intothe developing protein body and (4) fusion of large proteinbodies with one another at later stages of grain development.Of the four mechanisms described, the pinocytotic vesicles andfusion of protein bodies were the most frequent and consistentprocesses observed. Direct connections between rough endoplasmicreticulum (RER) and protein bodies were not observed. The resultssuggest a rôle for the Golgi apparatus in the initiationof protein bodies. Also, the lack of RER derived vesicles suggestsa soluble mode of secretion of storage proteins involved inthe enlargement of protein bodies. Triticum aestivum, wheat endosperm, protein bodies Golgi apparatus     

FINE STRUCTURE AND CYTOCHEMISTRY OF LILIUM POLLEN TUBES

Growth of the Lilium longiflorum pollen tube in vitro is restricted to a zone extending back 3–5 μ from the tip. Electron micrographs of cross and longitudinal thin sections of L. longiflorum and L. regale pollen tubes reveal that the cytoplasm of the nongrowing region of the tube contains an abundance of mitochondria, amyloplasts, Golgi bodies, endoplasmic reticulum, lipid bodies, and vesicles. In contrast, the growing tip is characterized by an abundance of vesicles and an absence of other cytoplasmic elements. The vesicles appear to be of 2 types. One is spherical, about 0.1 μ in diameter, stains strongly with phosphotungstic acid, apparently arises from the Golgi apparatus and appears to contribute to tube wall and plasmalemma formation. The other type is irregular in shape, 0.01-0.05 μ in diameter, stains strongly with lead hydroxide, and is of unknown origin and function. Cytochemical analysis indicates that the tips of L. longiflorum pollen tubes are singularly rich in ribonucleic acid, protein, and carbohydrate. These findings are discussed in relation to tube growth.     

Co-bombardment,integration and expression of rice chitinase and thaumatin-like protein genes in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> cv. Conlon)

Pathogenesis-related (PR) proteins associated with degradation of structural components of pathogenic filamentous fungi were overexpressed in the two-rowed malting barley (Hordeum vulgare L.) cultivar Conlon. Transgenes were introduced by co-bombardment with two plasmids, one carrying a rice (Oryza sativa L.) chitinase gene (chi11) and another carrying a rice thaumatin-like protein gene (tlp). Each gene was under the control of the maize ubiquitin (Ubi1) promoter. Fifty-eight primary transformants from three independent transformation events were regenerated. T₁ plants with high rice chi11 and tlp protein expression levels were advanced to identify T₂ homozygotes by herbicide spray and subjected to further molecular analyses. T₃ progeny from one event (E2) had stable integration and expression of the rice chi11 and tlp while those from the other events (E1 and E3) showed stable integration only of tlp. The successful production of these lines overexpressing the antifungal chi and tlp proteins provides materials to test the effects of these genes on a variety of fungal diseases that attack barley and to serve as potential additional sources of disease resistance.     

Immunocytochemical evidence for the involvement of golgi apparatus in the deposition of seed lectin ofBauhinia purpurea (Leguminosae)

Summary The seed lectin of the tree legume,Bauhinia purpurea alba, was localized by electron microscopic immunocytochemistry. The pattern of lectin deposition and site of intracellular localization was examined in mid- to late-maturation seeds. The seed tissue was embedded in Lowicryl K4M, the use of which with seed tissues is discussed. Immunocytochemical labeling was accomplished with colloidal gold coupled to a second antibody. The immunocytochemical reaction was specific and sensitive. Protein bodies, Golgi apparatus and Golgi secretion vesicles were densely labeled. Golgi apparatus was oriented such that Golgi secretion vesicles were in close proximity to the protein bodies. The entire Golgi apparatus was labeled with no concentration gradient across the Golgi stack. These observations indicate that the final site of lectin deposition is the protein body, and that the Golgi apparatus plays an essential role in the deposition process.     

Formation of Protein Bodies in the Starchy Endosperm of Rice (Oryza sativa L.): A Re-investigation

The mechanism of protein body formation in the starchy endospermis described for two rice varieties, one of normal protein contentand the other high in protein. Three types of protein bodieswere found in both rices. While each protein body type was depositeddifferently, the mechanism of formation for the same type inthe two varieties was analogous. First secreted was the largespherical protein body. It was deposited within rough endoplasmicreticulum, had a dense centre and a concentric ring appearance,and was bounded by a single membrane. The second protein bodyto form was crystalline, was found in vacuoles, and was secretedvia the Golgi apparatus. The third type, the small sphericalprotein body, was secreted late in development, was depositedin vesicular rough endoplasmic reticulum, and lacked dense centresand concentric rings. The high protein rice had a thicker sub-aleuronelayer than the normal protein rice. Oryza sativa L., rice, protein bodies, starchy endosperm, ultrastructure     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins

A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, K_d and B_max values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.     

Coated Vesicles Are Involved in the Transport of Storage Proteins during Seed Development in Pisum sativum L

During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin-coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constitutents. A 23,000 M_r polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including α-mannosidase, α-galactosidase, and β-N-acetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development.     

Subcellular localization of the red-absorbing form of phytochrome by immunocytochemistry

Summary An immunocytochemical technique was used to localize the red-absorbing form of phytochrome at the light- or electron-microscope level in etiolated barley (Hordeum vulgare L.) coleoptile tip, rice (Oryza sativa L.) coleoptilar node, maize (Zea mays L.) coleoptile tip, rye (Secale cereale L.) coleoptile tip and coleoptilar node, and oat (Avena sativa L.) root cap. Staining for phytochrome in the cells was found to be generally distributed throughout the cytoplasm. In addition, barley also showed staining around the periphery of vesicles, and rice showed staining in numerous discrete regions in the cytoplasm. Electron-microscopic localization studies of the nodal region of rye and the root cap of oat indicate staining associated with the nuclear membrane and with the interior of mitochondria and amyloplasts as well as general staining like that observed with the light microscope. Cells of the coleoptile tip of maize were unusual in having heavy staining associated with amyloplasts only.Abbreviations DAB 3,3-diaminobenzidine - PAP peroxidase-antiperoxidase complex - Pr red-absorbing form of phytochrome - Pfr far-red-absorbing form of phytochrome     

The Golgi apparatus mediates the transport of phytohemagglutinin to the protein bodies in bean cotyledons

When developing cotyledons of Phaseolus vulgaris L. were labeled with [³H]fucose, fucose-labeled phytohemagglutinin (PHA) was found in organelles with average densities of 1.13 g cm^-3 and 1.22 g cm^-3. The position of these organelles on isopycnic sucrose gradients was independent of the presence of MgCl₂ and ethylenediaminetetraacetate in the media, indicating that the fucose-labeled PHA was not associated with the rough endoplasmic reticulum (ER). The organelles with a density of 1.13 g cm^-3 were identified as membranes of the Golgi apparatus on the basis of the similarity of their sedimentation properties and those of the Golgi marker enzyme, inosine diphosphatase, in both isopycnic and rate-zonal sucrose gradients. The organelles with a density of 1.22 g cm^-3 were identified as small (0.1–0.4 μm), electron-dense vesicles with a protein content similar to that of the protein bodies. Pulsechase experiments with [³H]fucose indicated that fucose-labeled PHA first appeared in the Golgi-apparatus-derived membranes and later in the dense vesicles. Fucose-labeled PHA chased out of the Golgi apparatus first, then out of the dense vesicles, and accumulated in the soluble portion of the homogenate which contained the contents of the broken protein bodies. Fucose-labeled PHA chased out of the two types of organelles with a t _1/2 of 20–30 min, a rate three to four times faster than newly synthesized PHA chases out of the bulk of the ER (Chrispeels, M.J., Bollini, R., 1982, Plant Physiol. 70, 1425–1428). This result indicates that the Golgi apparatus is a much smaller compartment than the ER in the storage parenchyma cells. The sodium ionophore, monensin, which interferes with the function of the Golgi apparatus of animal cells, blocks the biosynthesis and—or transport of fucose- and galactose-labeled macromolecules to the cotyledon cell walls. Monensin also blocks the transport of labeled PHA out of the Golgi apparatus and into the protein bodies. These results provide the first biochemical evidence that a specific storage protein which accumulates in seeds is modified in, and passes through, the Golgi apparatus on its way to the protein bodies.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

Chilling Sensitivity in Oryza sativa: The Role of Protein Phosphorylation in Protection against Photoinhibition

The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L.). Brief exposure to chilling temperatures (0-15°C, 10 min) did not cause a significant difference in photosynthetic O₂ evolution capacity in vivo between rice and barley. Analysis of in vivo chlorophyll fluorescence in chilling-sensitive rice suggests that low temperatures cause an increased reduction of the plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction centers. Analysis of ³²P incorporation into thylakoid proteins both in vivo and in vitro demonstrated that chilling temperature inhibited protein phosphorylation in rice, but not in barley. Low temperature (77 K) fluorescence analysis of isolated thylakoid membranes indicated that state I to state II transitions occurred in barley, but not in rice subjected to chilling temperatures. These observations suggest that protein phosphorylation may play an important role in protection against photoinhibition caused by exposure to chilling temperatures.     

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

Immunocytochemical Localization of Wheat Storage Proteins in Endosperm Cells 30 Days After Anthesis

Antisera against seven different wheat (Triticum aestivum L.)storage protein subfractions were characterized using (1) ELISAwith gliadins and low- and high-molecular weight glutenin subunitsand (2) electrophoresis (SDS-PAGE and acidic buffer PAGE) andimmunoblotting. The specificities of these antisera (polyclonalantibodies) and 13 monoclonal antibodies covered various patternsof reactivity with alpha-, beta-, gamma- and omega-gliadinsand low- and high-molecular weight glutenins. The antisera andantibodies were applied to ultrathin sections of wheat endospermtissue, from kernels fixed 30 d after anthesis, and were detectedby secondary antibodies tagged with either 5 or 15 nm gold particlesusing transmission electron microscopy. Labelling was denserwhen the small gold particles were used but irrespective ofgold particle size, labelling of polyclonal antisera predominatedwhen the endosperm cells were subjected to both mono- and polyclonalantibodies. Each of the antisera and monoclonal antibodies thatlabelled the protein bodies, labelled them more or less uniformly.This indicates that only one kind of protein body, containingall gliadin and glutenin subfractions, exists during this stageof grain development. Electron-dense globular inclusions foundin many protein bodies were not labelled. Label was also foundon protein-like material present in the lumen of the rough endoplasmicreticulum and on vesicles of the Golgi apparatus. Thus concentrationof storage proteins takes place both at the site of synthesis,the lumen of the rough endoplasmic reticulum, and at the siteof processing and transport, the vesicles of the Golgi apparatus.Fusions between these proteinaceous materials give rise to largerprotein bodies and ultimately to the protein matrix. Key words: Wheat, immunocytochemistry, protein bodies, rough endoplasmic reticulum, Golgi apparatus     

One Novel Mitochondrial Citrate Synthase from <Emphasis Type="Italic">Oryza sativa</Emphasis> L. can Enhance Aluminum Tolerance in Transgenic Tobacco

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibitted increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

Elektronenmikroskopische Untersuchungen von Differenzierungsvorgängen bei Moosen

Summary In meristematic cells of the gemma of Riella helicophylla and in young bud cells from the protonema of Funaria hygrometrica the cell plate is formed by fusion of small vesicles originating from the Golgi apparatus. These spherical vesicles of about 0.1 m diameter have an electron dense centre, probably consisting of pectic substances or their precursors. The endoplasmic reticulum producing multivesicular bodies participate in cell plate formation too. Another cytoplasmic component forming the cell plate are coated vesicles, the origin of which is the Golgi apparatus and perhaps also the endoplasmic reticulum. In view of these observations the question of whether the endoplasmic reticulum or the Golgi apparatus forms the cell plate must be answered in this way: both endoplasmic reticulum and Golgi apparatus supply material for growth of the cell plate. Multivesicular bodies, coated vesicles and other small vesicles of unknown nature participate in the formation of the primary wall.

---

---

Zum Teil finanziert mit Sondermitteln des Landes Niedersachsen an Prof. Dr. M. Bopp.     

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