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     

Accumulation of Metabolites during Seed Development in Trifolium repens L.

Metabolite deposition during seed development was examined histochemicallyin Trifolium repens by light- and fluorescence microscopy. Allendosperm haustorium at the chalazal pole of the embryo sacand wall protrusions in cell walls of the suspensor and theembryo sac suggest that transfer of metabolites from maternalto offspring tissue takes place primarily at these sites. Thisis further supported by prominent cutinization of the interpolarregion of the embryo sac wall, accumulation of starch in integumentaltissue at the embryo sac poles, and breakdown of interpolarendothelial cells. Decomposition of osteosclereid starch isfollowed by accumulation in the cellular endosperm and subsequentlyin the embryo parallel to endosperm degradation. The starchaccumulates gradually inward from the subepidermal cells ofthe embryo to the stele. Protein bodies are formed in the vacuolesalong the tonoplast, later to be cut off in vesicles releasedinto the cytoplasm. At maturity the embryo is packed with proteinand starch, but without lipid reserves. Phytin is observed inthe protein bodies. The mature embryo is surrounded by a proteinand starch containing aleurone layer which originates from theendosperm.Copyright 1994, 1999 Academic Press White clover, protein, starch, cuticle, embryo sac wall     

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.     

花生胚乳细胞化的超微结构观察

花生（ＡｒａｃｈｉｓｈｙｐｏｇｅａｅＬ．）心形胚期的胚乳游离核多瓣裂,或具长尾状结构。胚乳细胞质内有大量线粒体、质体、高尔基体、小泡及少量内质网。中央细胞壁有壁内突。球胚及心形胚期常见胚乳瘤。心形胚晚期,胚乳开始细胞化,胚乳细胞壁形成有３种方式,分别存在于不同的胚珠中：（１）从胚囊壁产生自由生长壁形成初始垂周壁,具有明显的电子密度深的中层,其生长主要靠末端的高尔基体小泡及内质网囊泡的融合。两相邻的自由生长壁末端或其分枝末端相连形成胚乳细胞。（２）核有丝分裂后产生细胞板,细胞板向外扩展并可分枝。间期的非姊妹核间也观察到形成了细胞板。小泡与微管参与细胞板的扩展,高尔基体和内质网是小泡的主要来源。细胞板的扩展末端相互连接,形成胚乳细胞的前身。小泡继续加入细胞板的组成,以后形成胚乳细胞壁。（３）胚乳细胞质中,出现一些比较大的不规则形的片段性泡状结构,它们可能来源于高尔基体小泡,这些片段性泡状结构随机相连形成细胞壁,未见微管参与。胚乳细胞外切向壁及经向壁上有壁内突。     

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     

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     

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     

THE PRESENCE OF PROTEASE-DIGESTIBLE MATERIAL IN GOLGI VESICLES DURING ENDOSPERM DEVELOPMENT OF SELECTED CEREALS

The presence of dictyosomes secreting densely stained vesicles throughout endosperm protein body formation was confirmed for four cereals (rice, Oryza sativa L.; hard red winter wheat, Triticum aestivum L.; winter feed barley and spring malting barley, Hordeum vulgare L.; oats, Avena sativa L.). The contents of the Golgi vesicles and protein bodies were digested with proteases for all cereals except rice. It was found in the case of rice that OsO₄ altered the proteins in the Golgi apparatus and protein bodies making them resistant to protease digestion. These results imply that the Golgi apparatus plays an important role in the concentration and transport of storage proteins into vacuoles.     

Development of basal endosperm transfer cells in Sorghum bicolor (L.) Moench and its relationship with caryopsis growth

During sorghum caryopsis development, endosperm epidermal cells near the basal main vascular bundle are specialized by depositing wall ingrowths, differentiating into basal endosperm transfer cells (BETCs). All the BETCs together compose the basal endosperm transfer layer (BETL). BETCs are the first cell type to become histologically differentiated during endosperm development. The initiation and subsequent development of BETCs shows the pattern of temporal and spatial gradient. The developmental process of BETL can be divided into four stages: initiation, differentiation, functional, and apoptosis stage. A placental sac full of nutrient solutions would emerge, enlarge, and eventually disappear between the outmost layer of BETL and nucellar cells during caryopsis development. BETCs have dense cytoplasm rich in mitochondria, lamellar rough endoplasmic reticulum, Golgi bodies, and their secretory vesicles. They show a series of typical characteristics of senescence such as nuclei distortion and subcellular organelle deterioration during their specialization. BETCs probably play an active role in nutrient transfer into the starchy endosperm and embryo. The occurrence, development, and apoptosis of BETCs are in close relation to the caryopsis growth and maturation especially the enrichment of endosperm and the growth of embryo. The timing when BETL is fully developed, composed of three to four layers in radial direction and 70 to 80 rows in tangential direction, consists with the timing when average daily gain of caryopsis dry weight reaches its maximum. It is conceivable that measures that delay the senescence and death of BETCs would help to increase the crop yield.     

Expression patterns of genes encoding endomembrane proteins support a reduced function of the Golgi in wheat endosperm during the onset of storage protein deposition.

Wheat storage proteins are deposited in the vacuole of maturing endosperm cells by a novel pathway that is the result of protein body formation by the endoplasmic reticulum followed by autophagy into the central vacuole, bypassing the Golgi apparatus. This model predicts a reduced role of the Golgi in storage protein accumulation, which has been supported by electron microscopy observations. To study this issue further, wheat cDNAs encoding three distinct proteins of the endomembrane system were cloned and characterized. The proteins encoded were homologues (i) of the ER translocon component Sec61 alpha, (ii) the vacuolar sorting receptor BP-80 which is located in the Golgi and clathrin-coated prevacuole vesicles (CCV), and (iii) the Golgi COPI coatomer component COP alpha. During endosperm development, the levels of all three mRNAs were highest in young stages, before the onset of storage protein synthesis, and declined with seed maturation. However, the relative mRNA levels of BP-80/Sec61 alpha and the COP alpha/Sec61 alpha were lower during the onset of storage protein synthesis than at earlier stages of endosperm development. These results support previous studies, suggesting a reduced function of the Golgi apparatus in wheat storage protein transport and deposition.     

薏苡胚乳细胞化的超微结构观察

采用透射电镜对薏苡早期的胚乳细胞化进行了研究,在胚乳游离核时期,胚乳游离核及细胞质绕中央细胞分布,游离核间没有发现胚囊壁内突、成膜体等结构。胚乳细胞化过程中初始垂周壁形成过程如下：（１）胚乳细胞质中出现液泡,使细胞质和核向中央液泡推进：（２）一对相邻细胞核间液泡成对存在,且呈垂周分布,而且两液泡间的细胞质很狭窄;（３）在这狭窄的细胞质中出现成行排列的小泡;（４）小泡融合形成细胞板,细胞板悬于两液泡     

The fine structure of aleurone cells in the soybean seed coat

Summary The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.     

Seed structure and histochemistry in the palm Euterpe edulis

The Euterpe edulis embryo consists of a prominent single cotyledon, a very short radicle-hypocotyl axis and an epicotyl. The epicotyl is obliquely angled with respect to the cotyledon; consequently it corresponds to one of the two categories recognized for palm seeds by DeMason (1988 ). Parenchyma, protoderm and procambium can be distinguished on the basis of position and shape of their cells, which are highly vacuolated with one central vacuole and the cytoplasm restricted to a thin parietal layer. Initial cells from both apical meristems are also vacuolated but they have small vacuoles distributed around the nuclei. Silica occurs in cell walls of some protodermal cells. Raphides, silica bodies and tannins all occur occasionally in vacuoles, especially in the basal cotyledon region. Most embryo cells lack storage reserves and exhibit an active state, with numerous mitochondria, RER cisternae and Golgi apparatus, indicating a strategy of continuous development without the interposition, at maturity, of a dry state. The endosperm consists of living cells with very large nuclei and thickened cell walls. Similar to the endosperm of other studied palm species, their cells exhibit a quiescent appearance with lipid, protein, minerals (in the cytoplasm) and mannans (in the cell walls) as the insoluble storage reserves.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 145 , 445–453.     

Functional Anatomy of the Ovule in Broad Bean (Vicia faba L.): Ultrastructural Seed Development and Nutrient Pathways

Ovules of broad bean (Vicia faba L.) were studied to discloseultrastructural features, which can facilitate nutrient transportto the embryo sac from 10 d after pollination (DAP) to the matureseed. Fertilization occurs during the first 24 h after pollination.The endosperm is a coenocyte, which is eventually consumed bythe embryo. By 10 DAP the inner integument is degraded and theouter integument adjoins the embryo sac boundary. The heart-shapedembryo approaches the embryo sac boundary at two sites, whichhere are named contact zones. Small integument cells in theneighbourhood of the first formed contact zones become separatedby prominent intercellular spaces. A heterogenous scatteringmaterial, probably representing secretion products accumulatesin these spaces. By 14-16 DAP the integument exudate disappears,and the suspensor degenerates. As the contact zones increasein size, wall ingrowths form a bridging network in the narrowspace between the embryo sac boundary and the extra-embryonicpart of the endosperm wall. The epidermal cells of the embryoseparate adjacent to these zones, and develop conspicuous wallingrowths. At 20 DAP vacuoles showing various stages in formationof protein bodies appear in the cells of the embryo.Copyright1994, 1999 Academic Press Vicia faba, broad beans, ovule, seed, nutrient transport     

COTTON EMBRYOGENESIS: THE EARLY DEVELOPMENT OF THE FREE NUCLEAR ENDOSPERM

An electron microscope study was made of the central cell and the development of the free nuclear endosperm surrounding the zygote and synergids during the first three days after pollination. The cytoplasm of the central cell, concentrated around the partially-fused polar nuclei, contains many ribosomes, mitochondria and large, dense, starch-containing plastids, some dictyosomes and lipid bodies, and long, single cisternae of rough endoplasmic reticulum (RER) that frequently terminate in whorls. Dense, core-containing microbodies are closely associated with the RER. After fertilization the cytoplasm of the 2-and 4-nucleate endosperm shows an increase in number of dictyosomes, and in amount of RER which becomes stacked in arrays of parallel cisternae. Cup-shaped plastids are associated with many long, helical polysomes. Perinuclear aggregates of dense, granular material also appear after fertilization. Granular aggregates and helical polysomes disappear after the first few divisions of the primary endosperm nucleus. During the second and third days of development there is an increase in dictyosome number and RER proliferation, and endosperm nuclei become deeply lobed. Concurrently, there is a sharp decline in the starch and lipid reserves of the central cell and elaborate transfer walls are formed at the micropylar end of the embryo sac and on the outer surface of the degenerating synergid. The transfer walls contain groups of small, membrane-bound vesicles, and are associated with large numbers of mitochondria and with the smooth endoplasmic reticulum.     

Embryo and Endosperm Development in Isatis Tinctoria L. and the Histochemical Study of Its Storage Reserve

The ovule is anatropous and bitegmic. The nuceIlar cells have disorganized except the chalazal proliferating tissue. The curved embryo sac comprises an egg apparatus and a central cell with two palar nuclei and wall ingrowths on its micropylar lateral wall. The antipodal cells disappear. Embryo development is of the Onagrad type. The filament suspensor grows to a length of 785 μm and degenerats at tarpedo embryo stage. The basal cell produces wall ingrowths on the micropylar end wall and lateral wall. The cells of mature embryo contain many globular protein bodies, 2.5–7.5 μm in diameter, composed of high concentration of protein and phytin, insoluble polysaccharide and lipid. The cells, except procambium, also contain many small starch grains. Some secretory cavities scattered in the ground tissue have liquidlike granules composed of protein, ploysacchaide and lipid. Endosperm development follows the nuclear pattern. At the late heart embryo stage, the endosperm around the embryo and the upper suspensor and the peripheral endosperm of the basal region of the U-shaped embryo sac becomes cellular. The endosperm at micropylar and chalazal ends remains free nuclear phase until the late bended cotyledon stage. Wall ingrowths at both micropylar and chalazal end wall and lateral wall of the embryo sac become more massive during endosperm development. Wall ingrowths also occur on the outer walls of the outer layer endosperm cells at both ends and lateral region of the embryo sac. When the embryo matures, many layers of chalazal endosperm ceils including 2–4 layers of transfer cells, a few of micropylar endosperm cells and 1–5 layers of peripheral endosperm cells are present. The nutrients of the embryo and endosperm at different stages of development are also discussed.     

花椒球心胚及胚乳的发生和发育

对花椒珠心胚及胚乳的发生和发育过程进行了详细的细胞学及细胞学研究。主要研究结果如下;珠心胚发生前,有性胚囊发育过程中从大孢子发生到胚囊形成的各个阶段均可发生退化,退化频率５０％,未退化的胚囊发育成熟,成熟胚囊仅含卵器和两个极核。卵器最终退化,极核不经受精自发形成胚肥。当胚乳游离核达到１５或３２个时,最早的珠心胚原始细胞由靠近胚囊球孔端的珠心细胞分化形成。随着子房生长,多个原始细胞持续不断地从珠孔端     

小麦受精过程的细胞化学研究

小麦成熟胚囊卵细胞中存在较多围核分布的淀粉粒和少量散布的脂类颗粒;两个助细胞中积累很多脂类,未见有淀粉粒存在;中央细胞中存在中等量均匀分布的淀粉粒和脂类颗粒。受精时期,胚囊内各细胞中淀粉粒变化不大。精卵核融合时,卵细胞和中央细胞中的脂类分别存在一个积累高峰。合子与相应时期游离核胚乳中的脂类颗粒均较少。原胚初期,每个原胚细胞及胚乳原生质中均积累较多脂类。珠孔附近的内珠被细胞中脂类颗粒较多,并存在一个有规律的变化。在观察的所有发育时期的胚珠中,均未发现贮存蛋白质。胚珠中脂类的一系列变化可能与雌性细胞的营养、胚胎发育初期的养料及花粉管的定向生长等有关     

Developmental changes in the subcellular structure of the seeds of Atropa belladonna

Subcellular changes in the embryo and endosperm of Atropa belladonna were studied at four developmental stages. The endosperm cells turn to storage cells much earlier than those of the embryo, which matures later. Cells of the cotyledon and radicle are very similar in structure. The young cells contain large osmiophilic spherosomes. The cytoplasm is filled with ribosomes but dictyosomes are very rare. Some proplastids, containing starch, and mitochondria are present in the early developmental stages but do not occur in the dormant cells. During ripening, the vacuoles of the endosperm cells and embryo develop into protein bodies. They become filled with protein material without any recognisable transport mechanism. Protein bodies have several electron-translucent globoid cavities and the protein mass contains a roundish or crystalline body. This body does not stain with potassium iodide but with periodic acid Schiff-reagent and protein stains, indicating that it contains glycoproteins. The embryo and endosperm cells of ripe Atropa seeds are very similar and filled with protein bodies and small spherosomes.     

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.