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The aim of this work was to investigate whether the nuclear matrix could provide the nucleation sites for dispersed parental nucleolar components to form post-mitotic prenucleolar bodies (PNBs). For this purpose, nuclear matrices from asynchronous populations of onion cells were fractionated, and the distribution of the insoluble components of the nucleolar processing complexes in the matrices were analysed by fibrillarin immunolabelling. The ultra-structural organization of the nuclear matrix of cells from late telophase to late G1, corresponding to the period of nucleolar reassembly and activation, was also analysed. Our results demonstrate that PNBs are structural components of the telophasic nuclear matrix and that this structure provides recruitment and assembly sites for the components of the nucleolar processing machinery, and suggests that the telophasic matrix network is involved in the early steps of post-mitotic nucleologenesis.

The analysis of isolated nucleolar matrices from onion cells by light and electron microscopy, 2-D separation of proteins, and confocal microscopy has confirmed the existence of an organized nucleolar matrix with a complex protein composition to which are attached the insoluble processing complexes. In the present work, we present evidence from immunoblotting, immunofluorescence, immunogold labeling, and preferential cytochemical staining with bismuth salts that an insoluble fraction of the multifunctional protein nucleolin, is a component of the onion nucleolar matrix, and analyse its ultrastructural distribution in the described domains of the matrix.

We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60-65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken anti-lamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA. The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Intranucleolar DNA, including ribosomal DNA (rDNA), was localized in situ in proliferating onion cells under the electron microscope using an anti-DNA monoclonal antibody and a postembedding indirect immunogold procedure. In the interphase nucleolus of this species, characterized by a very high amount of rRNA genes, we found DNA concentrated mostly in fibrillar centres (FCs) and in the region of the dense fibrillar component (DFC) immediately surrounding them. Clusters of gold particles were frequently seen covering both of these structural components of the nucleolus at the same time. Moreover, the same technique, applied to transcriptionally arrested quiescent onion cells, showed the nucleolar DFC devoid of DNA. Also, in mitotic cells at telophase, the prenucleolar material, which has the same morphological and cytochemical features as the DFC, does not contain DNA. These data suggest the existence of at least two subcomponents of the DFC in the onion cell nucleolus, one associated with pre-rRNA synthesis, and the other, with further processing of transcripts, already released from the rDNA template. We conclude that the first subcomponent forms part of the "transition between FC and DFC", which is the in situ structural counterpart of pre-rRNA synthesis. This transition is morphologicaly sizeable in onion cells, because of their high number of rRNA genes and the large size of the DFC mass; however, it would be largely detectable in situ in other cell systems, where the whole DFC comprises only a thin layer and the amount of rDNA is considerably reduced.