Abstract
Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- DEAE:
-
diethylaminoethyl
- MAb:
-
monoclonal antibody
- NEPHGE:
-
nonequilibrium pH gradient electrophoresis
References
Aebi, U., Chon, J., Buhle, L., Gerace, L. (1986) The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323, 560–564
Aaronson, R.P., Blobel, G. (1975) Isolation of pore complex in association with a lamina. Proc. Natl. Acad. Sci. USA 72, 1007–1011
Berezney, R. (1980) Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction. J. Cell Biol. 85, 641–650
Berezney, R., Coffey, D.S. (1974) Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60, 1410–1417
Berezney, R., Coffey, D.S. (1977) Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J. Cell Biol. 73, 616–637
Beven, A., Guan, Y., Peart, J., Cooper, C., Shaw, P. (1991) Monoclonal antibodies to plant nuclear matrix reveal intermediate filament-related components within the nucleus. J. Cell Sci. 98, 293–302
Dougall, D.K., Wetherell, D.F. (1974) Storage of wild carrot cultures in the frozen state. Cryobiology 11, 410–415
Dwyer, N., Blobel, G. (1976) A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581–591
Fey, E.G., Krochmalnic, G., Penman, S. (1986a) The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNA-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J. Cell Biol. 102, 1654–1665
Fey, E.G., Ornelles, D.A., Penman S. (1986b) Association of RNA with the cytoskeleton and the nuclear matrix. J. Cell Sci. Suppl. 5, 99–119
Fisher, D.Z., Chaudhary, N., Blobel, G. (1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc. Natl. Acad. Sci. USA 83, 6450–6454
Franke, W.W. (1987) Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell 48, 3–4
Frearson, E.M., Power, J.B., Cocking, E.C. (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol. 33, 130–137
Frederick, S.E., Mangan, M.E., Carey, J.B., Gruber, P.J. (1992) Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization. Exp. Cell Res. 199, 213–222
Fujimura, T., Komamine, A., Matsumoto, H. (1980) Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture. Physiol. Plant. 49, 255–260
Galcheva-Gargova, Z.I., Marinova, E.I., Koleva, S.T. (1988) Isolation of nuclear shells from plant cells. Plant Cell Environ. 11, 819–825
Georgatos, S.D., Maroulakou, I., Blobel, G. (1989) Lamin A, lamin B and lamin B receptor analogues in yeast. J. Cell Biol. 108, 2069–2082
Gerace, L., Blum, A., Blobel, G. (1978) Immunochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction: interphase and mitotic distribution. J. Cell Biol. 79, 546–566
Ghosh, S., Dey, R. (1986) Nuclear matrix network in Allium cepa. Chromosoma 93, 429–434
Hancock, R., Hughes, M.E. (1982) Organization of DNA in the interphase nucleus. Biol. Cell 44, 201–212
Holt, G.D., Snow, C.M., Senior, A., Haltiwanger, R.S., Gerace, L., Hart, G.W. (1987) Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Biol. 104, 1157–1164
Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., Sato, C. (1988) Intermediate filament reconstitution in vitro: The role of phosphorylation on the assembly-disassembly of desmin. J. Biol. Chem. 263, 5970–5978
Inoue, M., Hosaka, M., Matsumoto, T. (1984) Antigenic characterization of a Japanese common strain of tobacco mosaic virus by monoclonal antibodies. Ann. Phytopath. Soc. Japan 50, 19–26
Jordan, E.G. (1984) Nucleolar nomenclature. J. Cell Sci. 67, 217–220
Kaufmann, S.H., Shaper, J.H. (1984) A subset of non-histone nuclear proteins reversibly stabilized by the sulfhydryl crosslinking reagent tetrathionate. Exp. Cell Res. 155, 477–495
Krachmarov, C., Stoilov, L., Zlatanova, J. (1991) Nuclear matrix from transcriptionally active and inactive plant cells. Plant Sci. 76, 35–41
Krohne, G., Benavente, R. (1986) The nuclear lamins: a multigene family of proteins in evolution and differentiation. Exp. Cell Res. 162, 1–10
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–684
Lebkowski, J.S., Laemmli, U.K. (1982) Non-histone proteins and long-range organization of HeLa interphase DNA. J. Mol. Biol. 156, 325–344
LeStourgeon, W.C., Beyer, A.L. (1977) The rapid isolation, highresolution electrophoretic characterization, and purification of nuclear proteins. Methods Cell Biol. 16, 387–406
Masuda, K., Takahashi, S., Nomura, K., Inoue, M (1991) A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures. Plant Cell Rep. 10, 329–333
McKeon, F.D., Kirschner, M.W., Caput, D. (1986) Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature 319, 463–468
Moreno Diaz de la Espina, S., Barthellemy, I., Cerezuela, M.A. (1991) Isolation and ultrastructural characterization of the residual nuclear matrix in a plant cell system. Chromosoma 100, 110–117
Murashige, T., Skoog, F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497
O'Farrell, P.Z., Goodman, H.M., O'Farrell, P.H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133–1142
Pruss, R.M., Mirsky, R., Raff, M.C., Thorpe, R., Dowding, A.J., Anderson, B.H. (1981) All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell 27, 419–428
Scofield, G.N., Beven, A.F., Shaw, P.J., Doonan, J.H. (1992) Identification and localization of a nucleoporin-like protein component of the plant nuclear matrix. Planta 187, 414–420
Snow, C.M., Senior, A., Gerace, L. (1987) Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 104, 1143–1156
Towbin, J., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Traub, P., Scherbarth, A., Willingale-Theune, J., Traub, U. (1988) Large scale co-isolation of vimentin and nuclear lamins from Ehrlich ascites tumor cells cultured in vitro. Prep. Biochem. 18, 381–404
Verheijen, R., van Venrooij, W., Ramaekers, F. (1988) The nuclear matrix: structure and composition. J. Cell Sci. 90, 11–36
Worman, H.J., Henriquez, R., Schnell, D.J., Newman, J.L., Georgatos, S.D., Blobel, G (1989) Identification of a lamin B-like protein in plant cell nuclei. J. Cell Biol. 109, 133a
Author information
Authors and Affiliations
Additional information
We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.
Rights and permissions
About this article
Cite this article
Masuda, K., Takahashi, S., Nomura, K. et al. Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.. Planta 191, 532–540 (1993). https://doi.org/10.1007/BF00195755
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00195755