Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

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。     

Protein accumulation in aleurone cells,sub-aleurone cells and the center starch endosperm of cereals

There are mainly three endosperm storage tissues in the cereal endosperm: aleurone cells, sub-aleurone cells and the center starch endosperm. The protein accumulation is very different in the three endosperm storage tissues. The aleurone cells accumulate protein in aleurone granules. The sub-aleurone cells and the center starch endosperm accumulate protein in endoplasmic reticulum-derived protein bodies and vacuolar protein bodies. Proteins are deposited in different patterns within different endosperm storage tissues probably because of the special storage properties of these tissues. There are several special genes and other molecular factors to mediate the protein accumulation in these tissues. Different proteins have distinct functions in the protein body formation and the protein interactions determine protein body assembly. There are both cooperation and competition relationships between protein, starch and lipid in the cereal endosperm. This paper reviews the latest investigations on protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm. Useful information will be supplied for future investigations on the cereal endosperm development.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase (spot 27) participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress (spots 8, 115, 118, 125, 130) and signal transduction (spots 7, 100, 108). According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm (spots 54, 61, 73) were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo (spots 27, 62, 122) were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

Alisma embryogenesis: The development and ultrastructure of the suspensor

Summary The development of the suspensor (consisting of a basal cell and a few chalazal cells) inAlisma plantagoaquatica andA. lanceolatum was investigated using cytochemical methods, light and electron microscopy. The basal cell becomes differentiated during the first three days of embryo development. As a result of endopolyploidization the volume of the nucleus rapidly increases, as does the quantity of chromatin it contains and the size of the nucleolus. As basal cell grows, its cytoplasm increases in volume and the number of organelles increase, and wall ingrowths begin to form on the walls at the micropylar pole of the cell. The full development and functioning of the suspensor occurs during the next three days. The enormous basal cell then attains its maximum degree of differentiation: its nucleus reaches a ploidy of 256n or 512n, the micropylar transfer wall is fully developed, as is the cytoplasm, rich in proteins, ribonucleic acids (RNA) and organelles, particularly dictyosomes and long cisternae of the rough endoplasmic reticulum. The chalazal suspensor cells joining the embryo proper to the basal cell also become differentiated. In the seven-day embryo the suspensor begins to degenerate which coincides with the cellularization of the endosperm at the micropylar pole of the embryo sac. The senescence of the suspensor involves the degradation of the nucleus, increasing cytoplasmic vacuolization, and a distinct decrease in protein and RNA content, first in the basal cell, then in the chalazal suspensor cells. Analysis of the development and ultrastructure of the basal suspensor cell suggests that it plays the role of an active metabolic transfer cell, translocating nutrients from the maternal tissues via the chalazal suspensor cells to the growing embryo proper.     

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm.

The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.     

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Thioredoxin and germinating barley: targets and protein redox changes

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.     

水稻胚囊超微结构的研究

水稻(Oryza sativa L.)胚囊成熟时,卵细胞的合点端无细胞壁,核居细胞中部,细胞器集中在核周围,液泡分散于细胞周边区域。助细胞珠孔端有丝状器,合点端无壁,核位于细胞中部贴壁处,细胞器主要分布在珠孔端,液泡主要分布在合点端。开花前不久,一个助细胞退化。中央细胞为大液泡所占,两个极核靠近卵器而部分融合,细胞器集中在极核周围和靠近卵器处,与珠心相接的胚囊壁上有发达的内突。反足细胞多个形成群体,其增殖主要依靠无丝分裂与壁的自由生长,反足细胞含丰富活跃的细胞器,与珠心相接的壁上有发达的内突。开花后6小时双受精已完成,合子和两个助细胞合点端均形成完整壁。合子中开始形成多聚核糖体、液泡减小。退化助细胞含花粉管释放的物质,其合点端迴抱合子。极核已分裂成数个胚乳游离核,中央细胞中细胞器呈活化状态。反足细胞仍在继续增殖。讨论了卵细胞的极性、助细胞的退化、卵器与中央细胞间界壁的变化、反足细胞的分裂特点等问题。     

Immunocytochemical localization of a wheat germ lysozyme in wheat embryo and coleoptile cells and cytochemical study of its interaction with the cell wall

Among several wheat (Triticum aestivum L.) germ proteins able to lyse Micrococcus lysodeikticus, one lysozyme (W1A) was purified by ion-exchange chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. Polyclonal antibodies against this lysozyme were raised in rabbits. The in situ localization of W1A lysozyme was achieved by the indirect protein A-gold technique. Large amounts of W1A lysozyme were found in cell walls whereas intercellular spaces, cytoplasm, and organelles were nearly free of labeling. Specificity of labeling was assessed with several controls. In an attempt to detect the presence of binding sites, W1A lysozyme was complexed to colloidal gold. Particles were specifically distributed in large amounts over wheat embryo and coleoptile cell walls. The absence of labeling over isolated coleoptile cell walls treated with 0.1 and 0.4 molar potassium hydroxide for hemicellulose extraction indicated that W1A lysozyme binding sites were probably of hemicellulosic nature.     

Storage reserves and cellular water in mature seeds of Araucaria angustifolia

Seed tissues of Araucaria angustifolia (Bertol.) Kuntze were investigated using histochemistry, transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis. Moisture content and water status in tissues were also evaluated. In the embryo, TEM studies revealed the presence of one to several central vacuoles and a peripheral layer of cytoplasm in cells from different tissues of the cotyledons and axis. In the cytoplasm, lipid bodies, starch grains, mitochondria and a nucleus are evident. In most tissues, vacuoles contain proteins, indicating that the storage proteins are highly hydrated. In cells of the root cap, proteins are stored in discrete protein bodies. Both protein storage vacuoles and discrete protein bodies have inclusions of crystal globoids. EDX analysis of globoids revealed the presence of P, K and Mg as the main constituents and traces of S, Ca and Fe. In the root and shoot meristems, deposits of phytoferritin are present in the stroma of proplastids. The gametophyte consists of cells characterized by relatively thin cell walls and one to several nuclei per cell. Protein and lipid bodies are present, although starch is the most conspicuous reserve. Immediately after shedding, moisture content is approximately 145% (dry weight) for the embryo and 95% (dry weight) for the gametophyte. Calorimetric studies reveal that axes and cotyledons have a very high content of freezable water, corresponding to types 5 and 4, i.e. dilute and concentrated (or capillary) solution, respectively. The results are discussed in relation to the behaviour of the species, which has been categorized as recalcitrant.  © 2002 The Linnean Society of London . Botanical Journal of the Linnean Society , 2002, 140 , 273−281.     

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.     

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     

Identification and immunocytochemical localization of α-galactosidase in resting and germinated date palm (Phoenix dactylifera L.) seeds

α-Galactosidases (EC 3.2.1.22) from resting and germinated date (Phoenix dactylifera L.) seeds were compared and localized using immunocytochemical methods. The enzyme was present in both the endosperm and embryo of resting seeds, in the endosperm undergoing digestion where the greatest specific activity was present, and in the haustorium of seedlings. The enzyme had a molecular mass of 140000 as determined by gel filtration and a pH optimum of 4.5. At least seven forms of the enzyme with isoelectric points ranging from 3.85 to 5.2 were detected in the haustorium whereas only four of these forms were present in the endosperm. The relative activity levels of the various forms also differed between the two tissues. On Western blots all enzyme forms were recognized by antibodies raised against mung-bean (Vigna radiata) α-galactosidase. Using immunogold techniques, label was shown to be present in the protein bodies of the resting embryo cells but to decrease in this organelle as the reserve protein was mobilized and to appear diffusely in the cytoplasm in subsequent stages. In resting endosperm cells, label occurred in the protein bodies and in a thin region of inner wall. In endosperm undergoing digestion, where different stages of protoplast and wall breakdown occurred, immunogold staining was localized in the flocculent contents of vacuoles which resulted from storageprotein breakdown, then dense staining occurred in the inner wall of cell cavities formed by the complete dissolution of the cytoplasm, and finally, staining was uniformly diffuse throughout the remaining endosperm wall adjacent to the haustorium surface. These observations indicate that the α-galactosidase present in cell walls of the date palm endosperm during mannan mobilization is not secreted by the haustorium but instead is probably a pregermination product stored mainly in the protein bodies of resting endosperm and is released to the wall following loss of membrane integrity.     

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [¹⁴C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, -amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.Abbreviations CIE Crossed immunoelectrophoresis - SDSPAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

The cytological responses of tomato-seedling cotyledons to chilling and the influence of membrane modifications upon these responses

Summary Tomato seedlings were exposed to 3 days of chilling at 5 °C in both the presence and absence of 5 mM ethanolamine. An array of pathological responses could be seen at both the light and electron microscope levels: apparent loss of cell turgor, vacuolization, reduction in the apparent volume of both the cytoplasm and the vacuolar protein bodies, apparent deposition of new material in the cell walls, general disorganization of organelles, especially plastids, and a general loss of cytoplasmic structure. Chilling injury was less pronounced in ethanolamine-treated cotyledonary tissues which appeared to have normal turgor and cell shape at the light microscope level and better preserved organelles at the ultrastructural level. Protection by ethanolamine at the ultrastructural level was most pronounced for the cytoplasm, the mitochondria, and the cell walls. Dead cells were seen with both treatments but were 2–3 times as numerous in the chilled-only tissue.     

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.     

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.