Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Abstract

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 xbere digested with DNase I in combination with RNase A, followed by extraction with 1 M NaC1. 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).

Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells

“…NMCP1 is predicted to represent a tripartite structure comprising head, rod, and tail domains. The rod domain is a region representing alpha helices with heptad repeats of apolar residues and extrapolated to form coiled-coils (Masuda et al 1993). In a broad range of plant species, genes related to NMCP1 have been identified in a database, and some NMCP1-like proteins are reported to localize exclusively in the nuclear periphery (Moriguchi et al 2005;Dittmer et al 2007).…”

“…In these approaches, the use of monoclonal antibodies raised against nuclear matrix proteins from D. carota (Masuda et al 1993) facilitated distinguishing NMCP2 from NMCP1. Unfortunately, in plants, integral inner NM protein that is usable as a marker of NM has not been identified; therefore, NM breakdown and reformation was pursued by changes in the endomembrane system after staining with DiOC 6 (Yang et al 1997;Yamada et al 2006), together with the distribution of an ER-resident marker protein, protein disulfide isomerase (Herman et al 1990).…”

“…The nuclear lamina is resistant to extraction with nonionic detergents and high salt solutions, and the nucleus of land plants is known to possess a structure resistant to these extractions (Moreno Díaz de la Espina et al 1991;Frederick et al 1992;Masuda et al 1993). Nevertheless, analyses of the complete genome sequences in some dicotyledonous and monocotyledonous plants have demonstrated that plants lack not only lamin genes but intermediate filament protein genes (Cohen et al 2001 and see Meier 2007 for a review); the consensus sequence that is shared in lamins and intermediatetype filament proteins is not found in proteins deduced from plant genomes.…”

Discovering the World of Plant Nuclear Proteins

“…Carrot NMCP1 localizes only to the nuclear periphery (Masuda et al 1993(Masuda et al , 1997. Four NMCP1-related proteins were characterized in Arabidopsis thaliana; LINC1 (little nuclei 1) localizes to the nuclear periphery, whereas LINC2 (Dittmer et al 2007) localizes to the nucleoplasm.…”

AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

“…Further purification of nuclei by Percoll gradient centrifugation and preparation of the nuclear matrix with lithium diiodosalicylate (either with or without heat stabilization) were performed as described previously (Minami et al, 1999). High-salt extraction of nuclear matrix was performed essentially as described by Masuda et al (1993). Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl 2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12ЊC for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 g/mL.…”