Nucleoli and nucleolus‐associated chromatin (NAC) of radicle cells have been three‐dimensionally reconstructed from serial ultrathin sections during early germination of Zea mays. As a preliminary, the effect of 5 methods of fixation on the ultrastructure of the active NAC were tested qualitatively and quantitatively. It appeared that paraformaldehyde best preserved the fibrillar centres (FCs) and was consequently used for the 3‐D reconstructions. In quiescent cells, the NAC forms either 2 short internal strands about 0.7 μm thick running within the nucleolus or 2 peripheral knobs of the same diameter. Whatever its morphology, the NAC was composed of one clear zone, i.e., secondary constriction (SC) of the nucleolar organizer region (NOR), and one electron‐opaque zone, i.e., heterochromatic segment (HS). During germination the NAC was always connected to the nuclear envelope (NE) by a bridge of dense chromatin. The NAC strands or knobs of the quiescent cells are likely to be the counterpart of the 2 NORs of this species. 10–12 hr after onset of germination, one or several networks of nucleolar vacuoles were formed within which the whole NAC was located. Chromatin fibers about 12 nm thick emerged from unfolding portions of the NAC within these “nucleolar chromatin dispersal vacuoles” (NCDV). At 24 hr, the NAC appeared as 2 dichotomous strands. Seventy‐two hr after germination both the stretching out and branching of the NAC were more pronounced. After 120 hr, the transcribing ribosomal genes for each NAC strand together with the newly synthesized RNP transcripts formed a layer of dense fibrillar component surrounding a thin axis which was composed mainly of pale material. Together these formed a typical plant nucleolonema.
Summry— Ag staining was applied on interphasic nucleoli of Zea mays root cells 120h after germination. We applied the two‐step Ag‐NOR staining technique to small root fragments and the one‐step technique to sections of Lowicryl‐embedded tissue. The small‐sized silver grains were mainly located in the dense fibrillar component (DFC). The unstained fibrillar centers (FCs) differed in their proteinic contents from the NOR (which is positively silver stained) and were not the interphasic NOR counterpart.
Spatial relationships between the internal nucleolus-associated chromatin (NAC) and the numerous nucleolar vacuoles that appear during early germination have been studied in nucleoli of quiescent (non-germinated) and early germinating embryos of Sinapis using serial sections. In quiescent non-vacuolated nucleoli, the transcriptionally inactive internal NAC is a short strand about 900 nm thick that in cross-section appears as heterogeneous fibrillar centres (FCs). At 4 and 6 h after germination one or several large networks of interconnected nucleolar vacuoles develop around the dispersing internal NAC. Clumps of dense chromatin are still present within the nucleolar vacuoles and are probably unfolding into deoxyribonucleoprotein (DNP) fibres (about 110 nm thick), which rapidly intrude within the nucleolar body and form thin chromatin threads. At 24 h after germination the internal NAC is more dispersed and forms, for its greatest part, a long thread (about 240 nm in diameter) wrapped up with a few dense fibrillar component, the whole forming the first outline of a nucleolonema. In cross-section most of the internal NAC appears as homogeneous FCs but short portions remain more condensed and appear as heterogeneous FCs always associated with a nucleolar vacuole. From 48 h the internal NAC is a longer thinner strand (about 160 nm in diameter), probably continuous and surrounded entirely by a homogeneous muff of dense fibrillar component, the whole forming a typical nucleolonema (about 950 nm thick) meandering throughout the nucleolus. Small amounts of the internal NAC still remain undispersed in the form of heterogeneous FCs associated with a nucleolar vacuole. The repeated association of nucleolar vacuoles and dispersing internal NAC suggests that they could play a role in chromatin dispersion and, or, activation by creating a favourable microenvironment.
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