Abstract
Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.
Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.
Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.
The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm3 (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bailey, C.J., Cobb, A., Boulter, D.: A cotyledon slice system for the electron autoradiographic study of the synthesis and intracellular transport of the seed storage protein of Vicia faba. Planta 95, 103–118 (1970)
Barker, R.D.J., Derbyshire, E., Yarwood, A., Boulter, D.: Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds, and their intracellular and cotyledonary distribution. Phytochemistry 15, 751–757 (1976)
Baumgartner, B., Tokuyasu, K.T., Chrispeels, M.J.: Localization of vicillin peptidohydrolase in the cotyledons of mung bean seedlings of immunofluorescence microscopy. J. Cell. Biol. 79, 10–19 (1978)
Beachy, R.N., Thompson, J.F., Madison, J.T.: Isolation of polyribosomes and messenger RNA active in in-vitro synthesis of soybean seed proteins. Plant Physiol. 61, 139–144 (1978)
Beevers, L., Poulson, R.: Protein synthesis in cotyledons of Pisum sativum L.I. Changes in cell-free amino acid incorporation capacity during seed development and maturation. Plant Physiol. 49, 476–491 (1972)
Bollini, R., Chrispeels, M.J.: Characterization and subcellular localization of vicilin and phytohemagglutinin, the two major reserve proteins of Phaseolus vulgaris L. Planta 142, 291–298 (1978)
Bonner, W.M., Laskey, R.A.: A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88 (1974)
Briarty, L.G.: Stereology in seed development studies: some preliminary work. Caryologia 25, Suppl, 289–301 (1973)
Burr, B., Burr, F.A.: Zem synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc. Natl. Acad. Sci. USA 73, 515–519 (1976)
Ericson, M.C., Chrispeels, M.J.: The carbohydrate moiety of mung bean vicilin. Aust. J. Plant. Physiol. 3, 763–769 (1976)
Ericson, M.C., Delmer, D.P.: Glycoprotein synthesis in plants. I. Role of lipid intermediates. Plant Physiol. 59, 341–347 (1977)
Ericson, M.E., Delmer, D.P.: Glycoprotein synthesis in plants. III. Interaction between UDP-N-acetylglucosamine and GDP-mannose as substrates. Plant Physiol 61, 819–823 (1978)
Evans, M., Croy, R.R.D., Hutchinson, P., Boulter, D., Payne, P.I., Gordon, M.E.: Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.). Planta 144, 455–462 (1979)
Felsted, R.I., Leavitt, R.D., Bachur, N.R.: Purification of the phytohemagglutinin family of proteins from red kidney beans (Phaseolus vulgaris) by affinity chromatography. Biochim. Biophys. Acta 405, 72–81 (1975)
Fox, J.E., Pratt, H.M., Shewry, P.R., Miflin, B.J.: The in-vitro synthesis of hordeins with polysomes from normal and high lysine varieties of barley. In: Acides Nucléiques et Synthèse des Protéines chez les Végétaux, pp. 501–509, Bogorad, L., Weil, J.H., eds. Colloq. Intern. CNRS No. 261, Paris 1976
Gilkes, N.R., Herman, E.M., Chrispeels, M.J.: Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings. Plant Physiol (in press)
Grabar, P., Williams, C.A.: Methode permettant l'étude conjugée des propriétés électrophoretiques et immunochimiques d'un melange de proteines; application au sérum sanguin. Biochim. Biophys. Acta 10, 193–194 (1955)
Higgins, T.J.V., Spencer, D.: Cell-free synthesis of pea seed proteins. Plant Physiol. 60, 655–661 (1977)
Kessler, S.W.: Rapid isolation of antigens from cells with a staphylococcal protein-A-antibody absorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115, 1617–1624 (1975)
Khoo, V., Wolf, J.J.: Origin and development of protein granules in maize endosperm. Am. J. Bot. 57, 1042–1050 (1970)
Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)
Larkins, B.A., Dalby, A.: In-vitro synthesis of zein-like protein by maize polyribosomes. Biochem. Biophys. Res. Commun. 66, 1048–1054 (1975)
Larkins, B.A., Davies, E.: Polysomes from peas. V. An attempt to characterize the total free and membrane-bound polysomal population. Plant Physiol. 55, 749–756 (1975)
Larkins, B.A., Hurkman, W.J.: Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol. 62, 256–263 (1978)
Leavitt, R.D., Felsted, R.D., Bachur, N.R.: Biological and biochemical properties of Phaseolus vulgaris isolectins. J. Biol. Chem. 252, 2961–2966 (1977)
Lord, J.M., Kagawa, T., Moore, T.S., Beevers, H.: Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J. Cell Biol. 68, 11–29 (1973)
Lowry, D.M., Rosebrough, M.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)
Luthe, D.S., Peterson, D.M.: Cell free synthesis of globulin by developing oat (Avena sativa L.) seeds. Plant Physiol. 59, 836–841 (1977)
Manen, J.F., Miège, M.N.: Purification et caractèrisation des lectines isolées dans les albumines et les globulines de Phaseolus vulgaris. Physiol. Vég. 15, 163–173 (1977)
McLeester, R.C., Hall, T.C., Sun, S.M., Bliss, F.A.: Comparison of globulin proteins from Phaseolus vulgaris with those from Vicia faba. Phytochemistry 12, 85–93 (1973)
Nagahashi, J., Beevers, L.: Subcellular localization of glycosyl-transferases involved in glycoprotein biosynthesis in the cotyledons of Pisum sativum L. Plant Physiol. 61, 451–459 (1978)
Öpik, H.: Development of cotyledon cell structure in ripening Phaseolus vulgaris seeds. J. Exp. Bot. 19, 64–76 (1968)
Palade, G.E.: Intracellular aspects of the process of protein synthesis. Science 189, 347–358 (1975)
Phillipp, E.-I., Franke, W.W., Keenan, T.W., Stadler, J., Jarasch, E.-D.: Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue. J. Cell Biol. 68, 11–29 (1976)
Pusztai, A., Watt, W.B.: Glycoprotein II. The isolation and characterization of a major antigenic and non-haemaglutinating glycoprotein from Phaseolus vulgaris. Biochim. Biophys. Acta 207, 413–431 (1970)
Roman, R., Brooker, J.D., Seal, S.N., Marcus, A.: Inhibition of the transition of a 40S ribosome-Met-tRNA meti complex to an 80S ribosome-Met-tRNA meti complex by 7-methylguanosine-5′-phosphate. Nature 260, 359–360 (1976)
Sabatini, D.D., Kreibich, G.L.: Functional specialization of membrane-bound ribosomes in eukaryotic cells. In: The Enzymes of Biological Membranes, vol. 2, pp. 531–579, Martonosi, A., ed., New York, Plenum Publ. Corp. 1976
Sun, S.M., Buchbinder, B.U., Hall, T.C.: Cell-free synthesis of the major storage protein of the bean, Phaseolus vulgaris L. Plant Physiol. 56, 780–785 (1975)
Sun, S.M., McLeester, R.C., Bliss, F.A., Hall, T.C.: Reversible and irreversible dissociation of globulins from Phaseolus vulgaris seeds. J. Biol. Chem. 249, 2218–2221 (1974)
Sun, S.M., Mutschler, M.A., Bliss, F.A., Hall, T.C.: Protein synthesis and accumulation in bean cotyledons during growth. Plant Physiol. 61, 918–923 (1978)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bollini, R., Chrispeels, M.J. The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons. Planta 146, 487–501 (1979). https://doi.org/10.1007/BF00380865
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00380865