Protein Bodies Of Seeds Research Articles

The Unusual Extracellular Localization of Conglutin γ in Germinating Lupinus albus Seeds Rules out its Role as a Storage Protein

Summary Antibodies specific to conglutin γ, a putative storage glycoprotein of Lupinus albus seeds, and to one of its constituent subunits devoid of carbohydrate, have been used to localize the protein in mature and germinating seeds by light and electron microscopy. Conglutin γ is located within the protein bodies of mature seeds, but after 2 days of germination labelling is also observed in the extracellular spaces of the cotyledonary tissue. By 8 days after germination, when most of the protein bodies and their electron dense contents have disappeared, conglutin γ is found predominantly within the intercellular spaces. In parallel experiments labelling of a typical lupin storage protein, a legumin-like globulin, was shown to be confined to the protein bodies in both mature and germinating seeds, but not to be localized within the intercellular spaces. On the basis of these findings the role of conglutin γ as a storage protein is questioned.

The microstructure of food protein assemblies

Abstract Proteins occur as natural and engineered assemblies in foods. This paper focuses on the microstructural aspects of some protein assemblies and their relation to processing and functionality. Examples discussed include: (a) disassembly of protein bodies in seeds during aqueous extraction; (b) freeze‐texturization and gelation of muscle proteins; (c) formation of large protein aggregates during caking of fish hydrolysate powders; (d) transformation of globular to fibrous aggregates and the role of Tg during extrusion cooking; (e) formation of mixed and filled dairy gels; and (f) kinetics of aggregation of β‐lactoglobulin.

Ultrastructural and histochemical postfertilization megagametophyte and zygotic embryo development of white spruce (Picea glauca) emphasizing the deposition of seed storage products

Deposition of major storage substances in the megagametophyte and embryo of white spruce (Picea glauca (Moench) Voss) was studied ultrastructurally and histochemically during seed development. Lipid bodies appeared to be secreted by the smooth endoplasmic reticulum. In the megagametophyte they were deposited rapidly from the club-shaped embryo stage until the early organogenesis of the embryo. Major lipid accumulation in the embryo took place during rapid cotyledon development and simultaneously with the buildup of protein bodies. Formation of protein bodies in the megagametophyte and in the embryo was first detected approximately 6 and 29 days after fertilization, respectively. It is suggested that in the megagametophyte, this process proceeded by (i) deposition of amorphous protein clumps onto tonoplasts of subdividing vacuoles during early stages of protein body formation, (ii) fusion of small cytoplasmic vesicles possibly derived from the rough endoplasmic reticulum, and (iii) deposition of protein around dense, membrane-bound vesicles attached to tonoplasts. The third process was not observed in the embryo. During advanced formation, fusion of cytoplasmic vesicles into developing protein bodies was the only process observed in the megagametophyte and in the embryo. White spruce seed protein bodies contained a variable number of crystalloids and globoid cavities except in the embryo in which only single globoid cavities were observed. Key words: Picea, ultrastructure, histochemistry, megagametophyte, embryo, seed.

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco.

A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and deposition of coixin in seeds of Coix lacryma-jobi

The synthesis and assembly of prolamins into protein bodies of Coix lacryma-jobi seeds were investigated. Coixins, the Coix prolamins, are grouped into two distinct classes namely α- and γ-coixins. Alpha-coixins are constituted by four size classes, while γ-coixins comprise only one molecular weight class. SDS-PAGE and western blot analysis of prolamins extracted from endosperm during seed development showed that α-coixins are synthesized at earlier developmental stages than γ-coixin. In vitro translation of polysomal RNA attached to protein bodies isolated from mid-maturation endosperm showed that these polyribosomes are highly enriched in coixin messages. Polysomal RNAs isolated from all developmental stages were electrophoresed and probed with cDNA clones representing α- and γ-coixins. The results confirmed the earlier expression of α-coixins and also demonstrated that coixin RNA accumulation in the endosperm occurs mainly at seed mid maturation. Protein bodies isolated from immature endosperm contained all coixin components as determined by SDS-PAGE and western blot analyses. Immunocytochemical analysis by electron and light microscopy revealed that the coixin components are spread all over the protein bodies. The protein bodies are localized in the starchy endosperm cells filling the spaces left by the starch granules. They are surrounded by continuous membranes and are larger than the protein bodies described for maize.

Protein-storing vacuoles in inner bark and leaves of softwoods

The seasonal occurrence of protein-storage vacuoles in parenchyma cells of the inner bark and leaf tissues of seven softwood species was examined. Previously published results showed that these organelles often fill the phloem parenchyma cells of the inner bark tissues in overwintering hardwoods, whereas they are absent from this tissue during the summer. We hypothesize that the organelles are involved in the storage of reduced nitrogen during wintering, in a manner analogous to protein bodies of seeds. A survey of the phloem and cambial parenchyma tissues in six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and in one deciduous softwood species (Larix decidua) was conducted. There was a large variation in the degree and timing of protein-storage vacuole formation between the individual genera and species. The organelles were not seen in summer samples of inner bark tissues of any of the genera or species examined. Protein-storage vacuoles were common in the bark tissues of Pinus, Abies and Thuja, occasionally seen in Picea, and rarely found in Larix during the winter. One-year-old leaves were also examined, since in all but Larix they are overwintering structures and can act as potential sites of nitrogen storage. Protein-storage vacuoles were present in Pinus and Thuja leaf tissue in both summer and winter, in Abies during winter only, and were absent from Picea leaf tissue at all times. These results indicate that the formation of protein-storage vacuoles prior to overwintering is not a ubiquitous phenomenon in softwoods.

Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds

Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl4. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein - the 13S globulin - of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.

The protein-body proteins phytohemagglutinin and tonoplast intrinsic protein are targeted to vacuoles in leaves of transgenic tobacco

To demonstrate the relationship between protein-bodies in seeds and vacuoles in other tissues, we expressed the coding sequences of two bean (Phaseolus vulgaris L.) protein-body proteins in tobacco (Nicotiana tabacum L.). We chose phytohemagglutinin-L (PHA-L) and tonoplast intrinsic protein (TIP) as representatives of the protein-body contents and protein-body membrane, respectively. The location of the two proteins in the leaves of transgenic tobacco was examined by immunocytochemistry and in preparations of isolated vacuoles. Tonoplast intrinsic protein accumulates primarily in tonoplasts in tobacco leaves, whereas PHA is found exclusively in the vacuolar sap, showing that the signals that target proteins to protein-bodies and their limiting membranes in seeds are correctly recognized in leaves. This observation provides further evidence that proteinbodies of dicotyledonous seeds should be considered as protein-storage vacuoles.

Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles

Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.

Seasonally dependent formation of protein-storage vacuoles in the inner bark tissues of Salix microstachya

The inner bark tissues of temperate hardwoods often act in the temporary storage of reduced nitrogen as protein during the overwintering period. Electron microscopic studies reported here demonstrate the analogy between the protein-storage vacuoles of the inner bark tissues and protein bodies in seeds. Development of these organelles parallels that of protein body formation seen in many dicotyledonous seeds. Coincident with the synthesis and sequestering of specific proteins, the large central vacuoles of the phloem parenchyma cells are slowly replaced over a 3- to 4-week period with numerous smaller protein-storage vacuoles (protein bodies). These arise via the subdivision of the larger vacuole and subsequent filling of the smaller vacuoles with protein. During this process there is a proliferation of both free ribosomes and rough endoplasmic reticulum in the ground cytoplasm. Stacks of rough endoplasmic reticulum are present in the peripheral cytoplasm and surround the smaller vacuoles as proteinaceous material is deposited. Golgi complexes, although not numerous, are present in the ground cytoplasm during the filling of the protein storage vacuoles. Key words: protein-storage vacuoles, protein body development, Salix microstachya, hardening, nitrogen storage, dormancy onset.

An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF).

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.

Spherical organelles, analogous to seed protein bodies, fluctuate seasonally in parenchymatous cells of hardwoods

A light microscopic study of the inner bark tissues of 11 temperate hardwoods showed seasonal differences in the degree of vacuolization and in the type and amount of storage material within phloem parenchyma and vascular cambium cells. Throughout the summer these cells were highly vacuolated, and starch-containing plastids were often present. During the overwintering period the typical large, central vacuoles were replaced by small, spherical organelles approximately 0.5–7.0 μm in diameter. The contents of the spherical organelles stained positively for protein. These organelles are considered to be analogous to protein bodies of seeds and to play a role in the storage of nitrogen. This study confirms and extends previous reports of protein bodies within parenchymatous cells of overwintering temperate trees. It further demonstrates that the organelles occur seasonally and that the formation of protein bodies for overwintering storage is a common phenomenon.

Lectin-like proteins accumulate as fragmentation products in bean seed protein bodies

The gene sequence of the Phaseolus vulgaris L. seed lectin-like protein (LLP) and its precursor (∼ 40 kDa) which is associated with the endoplasmic reticulum has been described, while the molecular characteristics of the mature protein and its cellular localization are still unknown. Our data, obtained using antibodies raised against a fusion protein, which was produced in Escherichia coli and contained most of the LLP sequence, indicate that mature LLP accumulates in the protein bodies of cotyledon cells. LLP consists of several polypeptides in the M r range 15 000 to 20 000, which are a result of proteolytic fragmentation of the protein precursor.

Biogenesis of protein bodies by budding from vacuoles in developing pumpkin cotyledons

Vacuoles were isolated from pumpkin cotyledons at three developmental stages and judged to be pure by light microscopic inspection and marker enzyme assays. The time sequence of structural changes of vacuoles were examined by light microscopic inspection in parallel with their stainability with neutral red. Vacuoles isolated from the early stage of cotyledon development were heterogeneous in size (O=2–10 Μm) but stained uniformly with the dye. In contrast, vacuoles isolated from the middle stage were much larger (O=5–15 Μm), and there exist one to three cores, unstainable with neutral red, within a single vacuole. Electron microscopic observation confirms that vacuoles contain a few protein cores in cotyledon cells at the middle stage. Characteristically at this stage, it was observable that some large cores (O=4Μm) were budding from vacuoles. At the late stage, size of vacuoles becomes much smaller (O=6Μm), nearly equal to that of the protein bodies in dry seeds. Importantly, at this stage most of the volume of each vacuole was occupied by a single core, and only a small matrix space was stainable with neutral red. Suborganellar fractionation indicates that the vacuolar cores were identical to the crystalloids deposited in the protein bodies in dry seeds. Overall results strongly provide the evidence that one crystalloid buds from the vacuole during the later stage of seed maturation, giving rise to a protein body.

Immunocytochemical localization of the Lathyrus ochrus (L.) DC seed lectin in seeds and seedlings

Lathyrus ochrus (L.) DC lectin was found to be localized within the protein bodies of both the cotyledons and embryo axis of mature seeds, by using immunocytochemical-labelling techniques involving rabbit antibodies against lectin, followed by goat antibodies against rabbit immunoglobulins (IgG) either fluoresceine-labelled (light microscopy) or adsorbed on colloidal gold particles (electron microscopy). Deposition of lectin inside the protein bodies was studied during seed development, together with its disappearance associated with the protein bodies coalescence occurring during seed germination. In both cases, a parallel quantification of lectin in ripening seeds and seedlings was carried out by radial immunodiffusion with rabbit antibodies against lectin. Our failure to detect lectin in other parts of the plant during its life-cycle suggests that lectin remains associated only with the protein bodies of seeds and seedlings.

Occurrence of Globoid Crystals in Cotyledonary Protein Bodies of Pisum sativum as Influenced by Experimentally Induced Changes in Mg, Ca and K Contents of Seeds

In many species globoid crystals in protein bodies of seeds are very common while in other species they are rarely observed. A review of literature suggested that the balance between divalent (Mg2+ and Ca2+) and monovalent (K+) cations may be important in determining whether or not globoid crystals will form. To test this hypothesis, experiments were carried out to add Mg and Ca, or Ca alone, to pods developing on K-deficient pea plants. While it was possible to cause a reduction in K concentration and increases in Mg and Ca concentrations, any changes to the normal mineral storage pattern in pea cotyledons were remarkably small. In some treatments, statistically significant increases in the ratio (Mg + Ca)/K were obtained and the sample with the greatest increase was examined in detail by electron microscopy and energy dispersive X-ray analysis. In this sample globoid crystals were common, in contrast to their normally rare occurrence in pea cotyledonary tissue.

Protein bodies in umbelliferous seeds. III. Characterization of calcium-rich crystals

The chemical composition of the calcium-rich crystal inclusions present in the seed protein bodies of carrot (Daucus carota L. cv. Imperator 408), wild carrot (Daucus carota L.), caraway (Carum carvi L.), anise (Pimpinella anisum L.), dill (Anethum graveolens L.), celery (Apium graveolens L. cv. Tall Utah), fennel (Foeniculum vulgare Mill.), parsnip (Pastinaca sativa L. cv. Hollow Crown), parsley (Petroselinum sativum L. cv. Moss Curled), and chervil (Anthriscus cerefolium L. cv. Curled) was determined. Using a variety of methods including X-ray diffraction, infrared spectroscopy, microincineration, energy dispersive X-ray analysis, solubility studies, and staining, the chemical composition of the calcium-rich crystal inclusions was identified as calcium oxalate.

Protein bodies in umbelliferous seeds. II. Elemental composition

The elemental composition of the protein bodies from several members of the Umbelliferae including carrot (Daucus carota L. cv. Imperator 408), wild carrot (Daucus carota L.), caraway (Carum carvi L.), anise (Pimpinella anisum L.), dill (Anethum graveolens L.), celery (Apium graveolens L. cv. Tall Utah), fennel (Foeniculum vulgare Mill.), parsnip (Pastinaca sativa L. cv. Hollow Crown), parsley (Petroselinum sativum L. cv. Moss Curled), and chervil (Anthriscus cerefolium L. cv. Curled) was determined using energy dispersive X-ray analysis. Globoid crystals in the endosperm usually contained P, K, and Mg or infrequently P, K, Mg, and Ca. In the embryos of carrot and caraway, P was always present with a combination of K, Mg, or Ca. Calcium was the only element detectable in the calcium-rich crystals. The proteinaceous matrix always contained S and K regardless of the inclusion present in the protein body.Quantitative determinations of P, Mg, K, and Ca are presented for all members studied except wild carrot, chervil, celery, and fennel. In all species analysed in this manner K or Ca were present in the highest amounts followed by P and Mg. Calcium-localization studies showed that most of the Ca is located in the endosperm and pericarp portions of the mericarp.

Protein bodies in umbelliferous seeds. I. Structure

The structure of the protein bodies from seeds of the family Umbelliferae has not been studied extensively since late in the 19th century. Using light and electron microscopy structural aspects of the protein bodies of carrot (Daucus carota L. cv. Imperator 408), wild carrot (Daucus carota L.), caraway (Carum carvi L.), anise (Pimpinella anisum L.), dill (Anethum graveolens L.), celery (Apium graveolens L. cv. Tall Utah), fennel (Foeniculum vulgare Mill), parsnip (Pastinaca sativa L. cv. Hollow Crown), parsley (Petroselinum sativum L. cv.Moss Curled), and chervil (Anthriscus cerefolium L. cv. Curled) were studied. Both endosperm and embryo protein bodies were investigated. Structurally, the protein bodies from all these genera were similar in that two types of protein bodies were found. One type consisted of a homogeneous, proteinaceous matrix and a number of variously sized, globoid crystal inclusions. The other type consisted of a homogeneous, proteinaceous matrix and either an individual or, more commonly, an aggregate of calcium-rich crystals arranged in a cluster usually termed a druse. Both types of protein bodies were never found in the same cell. Only globoid crystals were found in the embryo protein bodies. Protein bodies in the embryos were smaller, more numerous per cell, and often contained a flocculent, proteinaceous matrix.

α- D-mannosidase and α- D-galactosidase from protein bodies of Lupinus angustifolius cotyledons

Two forms of p-nitrophenyl α- D-mannosidase and p-nitrophenyl α- D-galactosidase were purified from the protein bodies of mature Lupinus angustifolius seeds. A MW of 300 000 was calculated for both α-mannosidase A and B with K m = 1.92 and 2.70 mM and activation energies of 10.9 and 10.8 kcal/mol, respectively. α-Galactosidase I and II had MWs of 70800 and 17000 with K m = 0.282 and 0.556 mM and activation energies 17.7 and 11.5 kcal/mol, respectively. The enzymes had acid pH optima and were inhibited by various metal ions, carbohydrates and glycoproteins. They were able to release free sugar from several putative natural substrate oligosaccharides and the Lupinus storage glycoprotein, α-conglutin.