Abstract
A highly sensitive immunoassay has been used for the detection of a major storage protein, legumin, in embryos of Pisum sativum L.; with this technique nanogram quantities could be measured. In the two varieties tested, legumin could be detected in embryos in vivo, when they had attained a fresh weight of 2·10-3 g and 3·10-3 g, respectively. Contrary to earlier claims, embryos cultured in vitro were shown to be capable of initiating legumin synthesis. This capacity to initiate legumin synthesis was confirmed by two-dimensional isoelectric focusing-electrophoresis and fluorography; embryos harvested before initiation of legumin synthesis and cultured in radioactive medium were shown to have synthesized legumin subunits. The amounts of legumin and total protein synthesized per unit fresh weight were consistently greater in vitro than in equivalent embryos grown in vivo.
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Abbreviations
- ELISA:
-
Enzyme-linked immunosorbent assay
- BSA:
-
bovine serum albumin
- IgG:
-
immunoglobulin
- SDS:
-
sodium dodecyl sulphate
- DSP:
-
Pisum cv. Dark Skinned Perfection
References
Bonner, W.M., Laskey, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Carasco, J.F., Croy, R., Derbyshire, E., Boulter, D. (1978) The isolation and characterization of the major polypeptides of the seed globulin of cowpea (Vigna unguiculata L. Walp) and their sequential synthesis in developing seeds. J. Exp. Bot. 29, 309–323
Casey, R (1979) Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds. Biochem. J. 177, 509–520
Clark, M.F., Adams, A.N. (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34, 475–483
Cullis, C.A. (1978) Chromatin-bound DNA-dependent RNA polymerase in developing pea cotyledons. Planta 144, 57–62
Davies, D.R. (1976) Variation in the storage proteins of peas in relation to sulphur amino-acid content. Euphytica 25, 717–724
Dudman, W.F., Millerd, A. (1975) Immunochemical behaviour of legumin and vicilin from Vicia faba: a survey of related proteins in the Leguminosae subfamily Faboideae. Biochem. Syst. Ecol. 3, 25–33
Dure, L.S. (1975) Seed formation. Annu. Rev. Plant Physiol. 26, 259–278
Guldager, P. (1978) Immunoelectrophoretic analysis of seed proteins from Pisum sativum L. Theor. Appl. Genet. 53, 241–250
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685
Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335–341
Lea, P.J., Hughes, J.S., Miflin, B.J. (1979) Glutamine- and asparagine-dependent protein synthesis in maturing legume cotyledons cultured in vitro. J. Exp. Bot. 30, 529–537
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275
Millerd, A., Spencer, D. (1974) Changes in RNA-synthesizing activity and template activity in nuclei from cotyledons of developing pea seeds. Aust. J. Plant Physiol. 1, 331–341
Millerd, A., Spencer, D., Dudman, W.F., Stiller, M. (1975) Growth of immature pea cotyledons in culture. Aust. J. Plant Physiol. 2, 51–59
Millerd, A., Thomson, J.A., Schroeder, H.E. (1978) Cotyledonary storage proteins in Pisum sativum. III. Patterns of accumulation during development. Aust. J. Plant Physiol. 5, 519–534
O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021
Scholz, G., Richter, J., Manteuffel, R. (1974) Studies on seed globulins from legumes. I. Separation and purification of legumin and vicilin from Vicia faba L. by zone precipitation. Biochem. Physiol. Pflanzen 166, 163–172
Stafford, A., Davies, D.R. (1979) The culture of immature pea embryos. Ann. Bot. 44, 315–321
Thompson, J.F., Madison, J.T., Muenster, A.E. (1977) In vitro culture of immature cotyledons of soya bean (Glycine max L. Merr.) Ann. Bot. 41, 29–39
Voller, A., Bartlett, A., Bedwell, D.E., Clark, M.F., Adams, A.N. (1976) The detection of viruses by enzyme-linked immunosorbent assay (ELISA). J. Gen. Virol. 33, 165–167
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Domoney, C., Davies, D.R. & Casey, R. The initiation of legumin synthesis in immature embryos of Pisum sativum L. grown in vivo and in vitro. Planta 149, 454–460 (1980). https://doi.org/10.1007/BF00385747
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DOI: https://doi.org/10.1007/BF00385747