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     

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.     

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.     

Pumpkin (Cucurbita sp.) seed globulin VII. Immunofluorescent study on protein bodies in ungerminated and germinating cotyledon cells

Protein bodies of pumpkin cotyledon cells were oval (about 10?7µm), and each was composed of a crystalloid, a globoidand proteinaceous matrix. They started to swell and fuse with1 day of imbibition. The proteinaceous matrix region expandedat the expense of crystalloids, and its electron density decreased.Finally, the protein bodies became central vacuoles includingmany small protein particles in about 8 days of germination. Fluorescent microscopy using antibodies raised against pumpkinseed globulin showed that fluorescence could not be observedin either protein bodies of ungerminated seeds or crystalloidsof germinating cotyledons, and only the proteinaceous matrixof germinating cotyledons became fluorescent. Probable causesof no fluorescence on crystalloids of seed globulin depositionwere considered. (Received November 9, 1979; )     

Anatomical and ultrastructural changes in aleurone and myrosin cells of Sinapis alba during germination

Summary The cells of the embryo of Sinapis alba L. include either aleurone or myrosin grains and all cells contain oil bodies. Aleurone grains and oil bodies are degraded during germination. The myrosin grains of each myrosin cell, on the other hand, gradually turn into one big vacuole containing the myrosin. Probably very little, if any, new myrosin is formed in the cotyledons and hypocotyl of the seedling after germination. No difference was found between aleurone and myrosin cells in the development of organelles. The cells of provascular bundles of the mature embryo contain different amounts of aleurone grains in different stages of development, and their organelles are more developed than those of all other cells.     

Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus.

Myrosinase (EC 3.2.3.1) is a glucosinolate-degrading enzyme mainly found in special idioblasts, myrosin cells, in Brassicaceae. This two-component system of secondary products and degradative enzymes is important in plant-insect interactions. Immunocytochemical analysis of Arabidopsis localized myrosinase exclusively to myrosin cells in the phloem parenchyma, whereas no myrosin cells were detected in the ground tissue. In Brassica napus, myrosinase could be detected in myrosin cells both in the phloem parenchyma and in the ground tissue. The myrosin cells were similar in Arabidopsis and B. napus and were found to be different from the companion cells and the glucosinolate-containing S-cells present in Arabidopsis. Confocal laser scanning immunomicroscopy analysis of myrosin cells in B. napus embryos showed that the myrosin grains constitute a continuous reticular system in the cell. These findings indicate that in the two species studied, initial cells creating the ground tissue have different potential for making idioblasts and suggest that the myrosinase-glucosinolate system has at least partly different functions. Several myrosinases in B. napus extracts are recovered in complex together with myrosinase-binding protein (MBP), and the localization of MBP was therefore studied in situ. The expression of MBP was highest in germinating seedlings of B. napus and was found in every cell except the myrosin cells of the ground tissue. Rapid disappearance of the MBP from the non-myrosin cells and emergence of MBP in the myrosin cells resulted in an apparent colocalization of MBP and myrosinase in 7-d-old seedlings.     

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.     

Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

Protein bodies of castor bean endosperm: isolation, fractionation, and the characterization of protein components

Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix. The protein bodies are apparently surrounded by a single membrane. The protein bodies were isolated by grinding and centrifuging in glycerol. Such isolated protein bodies were almost identical (after cytological fixation) to those observed in situ, except that the globoids were lost. However, membrane-like structures appear to have surrounded the globoids. Histochemical analysis of the isolated protein bodies showed that carbohydrates (glycoproteins) are localized only in the matrix region.     

Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.Abbreviations FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - PBS-T PBS with 0.5% (v/v) Tween-20 (polyoxyethylene sorbitane monolaurate) This work was supported by The Norwegian Research Council for Science and the Humanities. We wish to thank Professor Med. O.A. Haugen, Department of Pathology, University of Trondheim, Norway, for the skilful assistance provided regarding fixation and sectioning.     

Myrosin cells and dilated cisternae of the endoplasmic reticulum in the order Capparales

Ultrastructural results for different types of protein–rich cells in five families generally accepted as Capparalean (Brassicaceae, Capparaceae, Resedaceae, Tovariaceae and Moringaceae) and two others (Gyrostemonaceae and Bataceae) considered by some workers to be Capparalean, support their alignment in the order Capparales. The term myrosin cell is used for those protein–rich cells which are typically idio–blastic and characterized by a homogenous, granular proteinaceous material in the vacuole and a cytoplasm which is filled with an extensive rough endoplasmic reticulum. This idioblastic myrosin cell type is characteristic for the Brassicaceae, Capparaceae, Tovariaceae, Moringaceae and Gyrostemonaceae. The guard cells of stomata may appear as myrosin cells, in which case they are termed guard–cell myrosin cells; they are found in the Resedaceae, Tovariaceae and Bataceae. Other proteinaceous cells are those with protein–rich dilated cisternae (DC) of the endoplasmic reticulum (ER). One type is the organelle–like DC, utricular or irregular dilations of the ER, filled with protein and ribosome–studded. Utricular DC are characteristic for the Brassicaceae and Capparaceae. Another type of DC is represented by protein–containing vacuoles derived from the ER, protein–rich ER–dependent vacuoles; these are found in the Brassicaceae, Capparaceae, Resedaceae, Tovariaceae and Gyrostemonaceae. The myrosin cells and cells with protein–rich dilated cisternae are here regarded as taxonomic criteria for the order Capparales.     

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     

Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds

Proteolytic cleavage plays an important role in storage proteindeposition and reactivation in seeds. Precursor polypeptidesare processed by limited proteolysis to mature subunits of reserveproteins in storage tissue cells of developing seeds. Stepsof proteolytic processing are closely related to steps in intracellularprotein transfer through the endomembrane system and to thedeposition in the storage vacuole. In germinating seeds specialendopeptidases trigger storage protein breakdown by limitedproteolysis. The induced conformation changes of storage proteinsopen them to attack by additional endo- and exopeptidases whichdegrade the protein reserves completely. Proteases that catalyselimited cleavage or complete degradation are synthesized asprecursors which also undergo stepwise limited proteolysis whenthey are formed in cotyledons of developing or germinating seeds.In general, this processing transforms enzymatically inactiveproenzymes into active proteases. Different compartments participatein the processing steps. Many of the proteases are encoded bysmall multigene families. Different members of the correspondingprotease families seem to act during seed development and germination.Proteolytic processes that contribute to the molecular maturationand to the reactivation of storage proteins in dicotyledonousseeds seem to be controlled by (1) differential expression ofmembers of the protease-encoding gene families; (2) stepwiseprocessing and activation of protease precursor polypeptides;(3) transient differential compartmentation of precursors andmature polypeptides of proteases and storage proteins, respectively;and (4) interacting changes in storage protein structure andprotease action. The present knowledge on these processes isreviewed. Key words: Dicotyledons, seeds, storage proteins, proteolytic cleavage, proteases     

Water uptake and distribution in germinating lupine seeds studied by magnetic resonance imaging and NMR spectroscopy

Magnetic resonance imaging (MRI) was used to study temporal and spatial water uptake and distribution in germinating lupine ( Lupinus luteus L.) seeds. During 24 h of imbibition, water was unevenly distributed within the seed and some anatomical parts were more hydrated than others. Water entered the seed through the hilum and micropyle. The embryonic axis was the first to show hydration followed by seed coat and later cotyledons. The changes in water status were characterized by NMR spectroscopy. Analyses of T₂ relaxation times revealed a three-component water proton system (structural, intracellular and extracellular water) in germinating lupine seeds. The data on the components of transverse relaxation time studies indicated the complex exchange processes taking place between water components inside lupine seed over first 2.5 h of hydration, with a distinguished increase in structural water and decrease in other components. This speaks in favor of the high water-absorbing capacity of lupine seeds as related to high protein content. Germination was accompanied by swelling of protein bodies and changes in the organization of stored reserves with gradual disappearance of protein from the cells.     

Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans

Storage protein hydrolysis in the cotyledons of germinating mung beans (Phaseolus aureus Roxb.) was examined by histochemical techniques, and the autolytic capacity of isolated protein bodies was studied with biochemical methods. The localization of endopeptidase activity within the cotyledons was studied using an India ink-gelatin film technique. After 24 hours of imbibition, a low level of endopeptidase activity was found throughout the storage tissues of the cotyledons. A marked increase in activity was noted in cells farthest from the vascular bundles 48 to 60 hours after the start of imbibition. The decrease in storage protein followed the same spatial distribution starting in the cells farthest from the bundles. The cotyledons contain a population of cells in various stages of endopeptidase activity enhancement and storage protein degradation. A wave of endopeptidase activity moves progressively through the cotyledons towards the vascular bundles leaving behind areas devoid of stored reserves and low in endopeptidase activity. Observations on the morphology of protein bodies during germination indicate that the membrane surrounding them remains intact, while the reserves disappear. This result suggests that the protein bodies may be undergoing autolysis. To determine whether this may indeed be the case, protein bodies were isolated from the meal of mung bean seeds using an aqueous medium containing 80% glycerol. The protein body preparations and the cytoplasm were assayed for the presence of a number of enzymes which may be involved in the breakdown of the storage proteins. The protein bodies contained all, or nearly all, of the carboxypeptidase, α-mannosidase, N-acetyl-β-glucosaminidase, and caseolytic activity. The cytoplasm contained all, or most, of the leucine aminopeptidase and the trypsin-like activity (benzoyl arginine-p-nitroanalide as substrate). Incubation of the isolated protein bodies resulted in the release of amino acids. An analysis of the products of hydrolysis indicated that very little, if any, storage protein was being hydrolyzed during the incubation. Hydrolysis of the storage proteins present in the protein bodies was greatly accelerated by the addition of extracts from the cotyledons of 4-day-old seedlings. The results suggest that new enzymic activities not present in the protein bodies isolated from dry seeds must either be activated or synthesized and possibly added to the protein bodies before storage protein breakdown can begin.     

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.     

Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.     

The development of protein and oil bodies in the seed of Sinapis alba L.

Summary The cotyledons of Sinapis alba L. seed are the storage organs and first photosynthetic organs. The development of the cotyledon cell contents was studied using electron and light microscopy. From the heart shaped embryo (11 days from petal fall) to the mature seed, nine stages were examined.Both types of protein grains (designated aleurone grains and myrosin grains) were found to form within vacuoles, but the mode of protein accumulation differed with each type of grain.Oil bodies were apparent with the EM from 18 days onwards, but could not be seen to arise from the ER. They were granular in appearance at early stages, but later became electron transparent.     

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.