Protein Bodies from the Endosperm of Castor Bean: Subfractionation, Protein Components, Lectins, and Changes during Germination

Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm³, 1.30 g/cm³, and > 1.46 g/cm³, respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.     

Biogenesis of Protein Bodies in Embryonic Axes of Soybean Seeds (Glycine max. cv. Enrei)

Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.     

Protein bodies from the cotyledons of Cytisus scoparius L. (Link). Ultrastructure,isolation, and subunit composition of albumin,legumin and vicilin

The structure of protein bodies differs in the upper and lower parts of the cotyledons of mature seeds of Cytisus scoparius L. The palisade-mesophyll cells contain essentially homogeneous protein bodies, without globoids, but the protein bodies of the spongy-mesophyll cells are heterogeneous, with numerous globoids. Albumins, legumins and vicilins were selectively extracted from isolated protein bodies and their subunits separated by SDS-PAGE, under non-reducing and reducing conditions.Abbreviations SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The reserve proteins in the cells of mature cotyledons ofLupinus albus var.Lucky. I. Quantitative ultrastructural study of the protein bodies

Summary A quantitative ultrastructural study of the protein bodies of the lupin cotyledonary cells was performed to determine the protein content per cell. Two kinds of protein body were observed by transmission and scanning electron microscopy whatever the cellular type within the cotyledon. Some of these were conventional spherical structures, entirely filled with a dense protein matrix, others exhibited one large or several small light inclusions within the dense matrix. Even when a few light areas contained globoids, the majority remained of unknown nature, but could not be considered proteinaceous since they never reacted with specific protein dyes. The reserve protein content per cell was determined by image analysis on two seeds (L₁ and L₂) selected because they had a markedly different total protein content. The volume occupied by the dense matrix of the intracellular protein bodies was considered representative of the reserve protein quantity. The protein content per cell increased from the periphery to the centre of the cotyledon in the two seeds studied. The protein content per cell of the L₂ seed was generally found to be greater than the L₁ seed, in particular in the abaxial zone where it was markedly higher. The difference in total protein content of the two seeds was demonstrated to be primarily due to a differential alveolation of the protein bodies. These results will be used to study the relationship between the protein content of the cotyledonary cells and their nuclear DNA content.     

Degradation of Protein Bodies in Germinating Seeds of Brassica campestris L. var. sarson Prain

Histochemical investigations were carried out on dry and germinatingseeds of Brassica campestris var. sarson, to study the degradationof protein bodies with globoidal inclusions. During germination,a wave of protein body degradation sets in from the radicularend of the embryo, passing through the hypocotyl and shoot apex,and ending in the cotyledons. The digestion of protein bodiesis of the internal type. The various isolated parts of the embryoshowed a similar pattern of protein body digestion to that ofthe whole embryo, except that in some cells the globoids persistedeven after complete digestion of protein bodies; the rate wasfaster in the comparatively more expanded part of the isolatedorgans. No specific factor controlling the initiation of thewave of protein body digestion could be ascertained. Brassica campestris var. sarson, yellow sarson, seed, germination, protein bodies, degradation     

Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated γ′ chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.

After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

     

Recalcitrant seeds of horse chestnut lack protein bodies

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.), the bulk of protein in axial organs and cotyledons is accounted for by water-soluble proteins (albumins). In the cells of embryo, proteins are predominantly located in the cytosol, whereas the fraction of cell structures precipitate in the range from 1000 to 20000 g, accounting for only an insignificant part of total protein. Among the proteins of this fraction, there were no major components that could play a role of storage proteins. The aim of this work was to study deposition of protein in the vacuoles of cells of recalcitrant seeds of horse chestnut. Light microscopy and specific staining of protein and phytin did not detect protein bodies in the vacuoles of axial organs and cotyledons. Electron microscopy revealed traces of phytin in the vacuoles, but there were no formed globoids or considerable amount of protein therein. It is possible that precisely the absence of typical storage proteins and genetically determined desiccation in the course of maturation of recalcitrant seeds of horse chestnut stipulated preservation of the vacuoles that in mature recalcitrant seeds were not transformed into protein bodies.     

DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Subcellular Organization of Developing Embryos of Bidens cernua

Anatomical and submicroscopical changes in the cotyledons and radicles of Bidens cernua L. have been studied at five developmental stages. In the subcellular structure, these two plant organs are relatively similar but each developmental stage is characterized by a distinct fine structure. Protein bodies, which occupy the bulk of the cell in dormant embryos, develop as filling products of vacuoles. Ribosomes are seen abundantly at this stage, both in the nucleus and the plasma strands. Small vesicles which are the initials of globoids can be detected in the vacuoles even of rather young cells. They later associate at the periphery of protein masses secreted in the vacuoles. Many light globoids are seen in the protein bodies of mature cells. Some amyloplasts are present in the early developmental stages but not in the dormant cells. The endoplasmic reticulum becomes filled with osmiophilic storage fat, and later many spherosomes are seen between the protein bodies. Some osmiophilic material is also found in the intercellular spaces.     

Energy dispersive X-ray analysis of protein bodies in Protea compacta cotyledons

Summary The elemental composition of globoids in the protein bodies of Protea compacta cotyledons was studied by means of energy dispersive X-ray analysis. The globoid crystal was rich in phosphorus and calcium with lesser amounts of magnesium and potassium suggesting the presence of phytin in these structures.Abbreviation EDX energy dispersive X-ray analysis     

Protein Bodies from Buckwheat Seed Cotyledons: Isolation and Characteristics

Protein bodies were isolated from cotyledons of dry buckwheatseeds by homogenization in acetone with subsequent purificationin a 1.26 g cm^–3 to 1.53 g cm^–3 linear density gradientof a mixture of acetone with CCI₄. The purified fraction ofprotein bodies with globoids (PB I) had a buoyant density of1.48–1.51 g cm^–3 and was intact according to microscopicdata. Localization of hydrolytic enzymes and proteinase inhibitorsin the PB I fraction and in the fraction of the cytoplasm andmembrane material (CMM) was studied. It was shown that acidhydrolytic enzymes, such as aspartic proteinase, carboxypeptidase,acid phosphatase, -D-mannosidase and N-acetyl-ß-glucosaminidase,as well as chymotrypsin and trypsin inhibitors were predominantlylocalized in the PB I. BAPAase and SH-activated caseinase activitieswere equally distributed between the PB I and CMM fraction.The activities of leucine aminopeptidase and SH-independentcaseinase were noticeably predominant in the CMM fraction. Key words: Buckwheat, subcellular fractionation, protein bodies, hydrolases     

DEVELOPMENTAL CHANGES IN THE COTYLEDONS OF LUPINUS LUTEUS L. DURING AND AFTER GERMINATION

Cotyledons of Lupinus luteus were sampled from 1 to 21 days after sowing and processed for light microscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Length, width, and surface area of the cotyledons increased gradually until day 10. The thickness of the cotyledons increased from day 7 to day 12 and decreased thereafter. Morphometric analyses showed that the increase in length, width, and thickness of the cotyledon was due to cell expansion, and the decrease in thickness of the cotyledon was due to the decrease in the length of abaxial cells and in the total number of cells. Mesophyll development accompanied schizogenous and lysigenous air space formation. There were two structurally distinct types of protein bodies. Protein bodies in five to six layers of cells on the abaxial side did not contain globoids, while globoids were prominent in protein bodies in the center and adaxial side. Storage protein mobilization occurred first in the abaxial side of the cotyledon and proceeded toward the adaxial side. SDS-PAGE analysis showed that proteins ranged from 97 to 14 kD. High molecular weight α- and ß-conglutinins were more abundant in the abaxial region, whereas γ-conglutinin occurred in both abaxial and adaxial regions. In addition, there were five minor bands between 97 and 43 kD unique to abaxial region and five minor bands between 43 and 14 kD unique to adaxial region in the nonreduced protein profiles. The α- and ß-conglutinins began to decrease after imbibition and disappeared by day 7 after sowing. At this stage the subunits of ribulose-l,5-bisphosphate carboxylase/oxygenase and four new minor bands appeared.     

Influence of opaque-2 and floury-2 genes on formation of proteins in particulates of corn endosperm

Protein-rich subcellular particulates were isolated by zonal centrifugation from homogenates of endosperms of normal, opaque-2, and floury-2 mutant corn (Zea maize) kernels at different stages of development. In early stages the high lysine mutants vary from normal corn by greater production of a glutelin protein not associated with the matrix. This protein is high in lysine and may become a component of matrix glutelin at later stages of maturity. Differences in size and structure of zein-rich protein bodies were observed in the mutant strains when compared with normal corn. Enhanced production of nonmatrix glutelin as well as the reduction in synthesis of lysine-deficient zein is responsible for the improved lysine content of the mutant endosperms at early stages of development.     

The structure and composition of aleurone grains in the barley aleurone layer

Summary Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.     

棉花种子萌发过程中子叶蛋白体变化

对棉花种子萌发过程中子叶细胞内蛋白体的变化进行了详细的观察。干种子内存在仅由蛋白质基质组成无内含物的蛋白体,含有球状晶体的蛋白体和无含球状晶体和拟晶体的蛋白体。种子萌发过程中蛋白体逐渐液泡化,其降解方式可分为三种类型：（１）内部降解类型：（２）周边降解类型;（３）内部和周边同时降解类型。文中还一步进行了不同降解类型与酶的分布,蛋白体存在部位和萌发时间进程之间的关系。     

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.     

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.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.