Skip to main content
Log in

Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds

  • Published:
Planta Aims and scope Submit manuscript

Abstract

The synthesis of a major oil-body membrane brotein was studied in maturing soybean (Glycine max (L.) Merr.) cotyledons. The membrane contained four abundant proteins with apparent molecular mass (Mr) of 34000, 24000, 18000 and 17000. The Mr=24000 protein (mP 24) was selected for more detailed analysis. The protein was purified to apparent homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isolated from the gel by electroelution or chemical hydrolysis of gel crosslinks. It was then used to elicit rabbit antibodies which were judged to be specific when assayed by SDS-PAGE-immunoblot procedures. The mP 24 was localized in immature soybean cotyledon cells by indirect immunogold procedures on thin sections of Lowicryl- and LR-White-embedded tissue. Indirect labeling with the primary antiserum followed by colloidal gold-protein A showed specific labeling of the oil-body membrane and an absence of label on the other subcellular organelles including the endoplasmic reticulum (ER). Parallel tissue samples were studied by conventional transmission electron microscopy. Although segments of the ER were observed to be closely juxtaposed to the oil bodies, continuity between the two organelles was not observed. The synthesis of mP 24 was studied by in-vitro translation and in-vivo labeling with [3H]leucine followed by indirect immunoaffinity isolation of the labeled products. The SDS-PAGE fluorography results indicated that the primary translation product and the in-vivo synthesized protein have the same Mr, and this is also the same Mr as the protein in the mature membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DATD:

N N'-diallyltartardiamide

EM:

electron microscopy/scopic

ER:

endoplasmic reticulum

IgG:

immunoglobulin G

Mr :

apparent molecular mass

PBS:

phosphate-buffered saline

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TBS:

Trisbuffered saline

References

  • Adams, C.A., Norby, S.W., Rinne, R.W. (1983) Ontogeny of lipid bodies in developing soybean seeds. Crop Science 23, 757–759

    Google Scholar 

  • Baumgartner, B., Tokuyasu, K.T., Chrispeels, M.J. (1980) Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons. Planta 150, 419–425

    Google Scholar 

  • Bergfeld, R., Hong, Y.N., Kuhnl, T., Schopfer, P. (1978) Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L. Planta 143, 297–303

    Google Scholar 

  • Bos, T.J., Davis, A.R., Nayak, D.P. (1984) NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation. Proc. Natl. Acad. Sci. USA 81, 2327–2331

    PubMed  Google Scholar 

  • Brown, D.A., Simoni, R.D. (1984) Biogenesis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an integral glycoprotein of the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 81, 1674–1678

    PubMed  Google Scholar 

  • Chirgwin, J.M., Przybla, A.E., MacDonald, R.J., Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299

    PubMed  Google Scholar 

  • Craig, S., Goodchild, D.J. (1984) Periodate-acid treatment of sections permits on-grid immunogold localization of pea seed vicilin in ER and Golgi. Protoplasma 122, 35–44

    Google Scholar 

  • Frey-Wyssling, A., Grieshaber, E., Mühlethaler, K. (1963) Origin of spherosomes in plant cells. J. Ultrastruc. Res. 8, 506–516

    Google Scholar 

  • Fujiki, Y., Hubbard, A.L., Fowler, S., Lazarow, P.B. (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102

    Article  PubMed  Google Scholar 

  • Glisin, V., Crkvenjakov, R., Byus, C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 2633–2637

    PubMed  Google Scholar 

  • Greenwood, J., Chrispeels, M.J. (1985) Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and golgi complex of developing bean cotyledons. Planta 164, 295–302

    Google Scholar 

  • Herman, E.M., Shannon, L.M. (1984a) Immunocytochemical evidence for the involvement of golgi apparatus in the deposition of seed lectin of Bauhinia purpurea (Leguminosae). Protoplasma 121, 163–170

    Google Scholar 

  • Herman, E.M., Shannon, L.M. (1984b) Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons. Planta 161, 97–104

    Google Scholar 

  • Herman, E.M., Shannon, L.M. (1985) Accumulation and subcellular localization of α-galactosidase in developing soybean cotyledons. Plant Physiol. 77, 886–890

    Google Scholar 

  • Huang, A.H.C. (1984) Plant lipases. In: Lipases, pp. 420–442, Borgstrom, B., Brockman, H.L., eds. Elsevier Press, Amsterdam

    Google Scholar 

  • Jacks, T.J., Yatsu, L.Y., Altschul, A.M. (1967) Isolation and characterization of peanut spherosomes. Plant Physiol. 42, 585–597

    Google Scholar 

  • Kyte, J., Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132

    PubMed  Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

    PubMed  Google Scholar 

  • Lin, Y.H., Huang, A.H.C. (1983) Lipase in the lipid bodies of cotyledons of rape and mustard seedlings. Arch. Biochem. Biophys. 225, 360–369

    PubMed  Google Scholar 

  • Lin, Y.H., Wimer, L.T., Huang, A.H.C. (1983) Lipase in the lipid bodies of corn scutella during seedling growth. Plant Physiol. 73, 460–463

    Google Scholar 

  • Lingappa, V.R., Lingappa, J.R., Prasad, R., Ebner, K.E., Blobel, G. (1978) Coupled cell-free synthesis, segregation, and core-glycosylation of a secretory protein. Proc. Natl. Acad. Sci. USA 75, 2338–2342

    PubMed  Google Scholar 

  • Meinke, D.W., Chen, J., Beachy, R.N. (1981) Expression of storage-protein genes during soybean seed development. Planta 153, 130–139

    Google Scholar 

  • Moreau, R.A., Liu, K.D.F., Huang, A.H.C. (1980) Spherosomes of castor bean endosperm, membrane components, formation and degradation. Plant Physiol. 65, 1176–1180

    Google Scholar 

  • Muto, S., Beevers, H. (1974) Lipase activities in castor bean endosperm during germination. Plant Physiol. 54, 23–28

    Google Scholar 

  • Ory, R.L., Yatsu, L.Y., Kircher, H.W. (1968) Association of lipase activity with the spherosomes of Ricinus communis. Arch. Biochem. Biophys. 264, 255–264

    Google Scholar 

  • Palmiter, R.C., Gagnon, J., Walsh, K.A. (1978) Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. Proc. Natl. Acad. Sci. USA 75, 94–98

    PubMed  Google Scholar 

  • Qu, R., Wang, S., Lin, Y., Vance, V.B., Huang, A.H.C. (1986) Characteristics and biosynthesis of membrane proteins of lipid bodies in scutella of maize (Zea mays L.). Biochem. J. 235, 57–65

    PubMed  Google Scholar 

  • Schwarzenbach, A.M. (1971) Observations on spherosomal membranes. Cytobiologie 4, 145–147

    Google Scholar 

  • Sengupta, C., Deluca, V., Bailey, D.S., Verma, D.P.S. (1981) Post-translational processing of 7S and 11S components of soybean storage proteins. Plant Mol. Biol. 1, 19–34

    Google Scholar 

  • Slack, C.R., Bertaud, W.S., Shaw, B.D., Holland, R., Browse, J., Wright, H. (1980) Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum usitatissimum). Biochem. J. 190, 551–561

    PubMed  Google Scholar 

  • Slot, J.W., Geuze, H.J. (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38, 87–93

    PubMed  Google Scholar 

  • Spath, P.J., Koblet, H. (1979) Properties of SDS-polyacrylamide gels highly cross-linked with N,N'-diallyltartardiamide and the rapid isolation of macromolecules from the gel matrix. Anal. Biochem. 93, 275–285

    PubMed  Google Scholar 

  • Stobart, A.K., Styme, S., Högland, S. (1986) Safflower microsomes catalyse oil accumulation in vitro: A model system. Planta 169: 33–37

    Google Scholar 

  • Ternynck, T., Avrameas, S. (1972) Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde. FEBS Lett. 23, 24–28

    Article  PubMed  Google Scholar 

  • Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354

    PubMed  Google Scholar 

  • Wang, S.-m., Huang, A.H.C. (1987) Biosynthesis of lipase in the scutellum of maize kernal. J. Biol. Chem. 262, 2270–2274

    PubMed  Google Scholar 

  • Wanner, G., Formanek, H., Theimer, R.R. (1981) The ontogeny of lipid bodies (spherosomes) in plant cells. Ultrastructural evidence. Planta 151, 109–123

    Google Scholar 

  • Wanner, G., Theimer, R.R. (1978) Membranous appendices of spherosomes (oleosomes). Possible role in fat utilization in germinating oil seeds. Planta 140, 163–169

    Google Scholar 

  • Yatsu, L.Y., Jacks, T.J. (1972) Spherosome membranes. Half unit-membranes. Plant Physiol. 49, 937–943

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Herman, E.M. Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds. Planta 172, 336–345 (1987). https://doi.org/10.1007/BF00398662

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00398662

Key words

Navigation