An ultracytochemical study of nucleolar organization in meristematic plant cells (Allium porrum).

The interphase nucleolus in Allium porrum, as in many of the plant species studied so far, is highly heterogeneous in ultrastructure owing to the presence of coarse, contorted, thread-like structures, or nucleolonemata. Each nucleolonema appears to be sharply twisted and to give rise to a skein within the nucleolar mass. In order to characterize further these nucleolar components, a variety of cytochemical techniques were exploited. For that purpose, specimens were mostly fixed in 4% formaldehyde and stained in the block according to procedures known to reveal the presence of nucleic acids or proteins. Certain specimens were also digested with deoxyribonuclease, ribonuclease or proteinase K before staining. By staining with phosphotungstic acid or bismuth oxynitrate, the presence of a high concentration of proteins can be demonstrated within thin (0.15 micrometer), filamentous structures which are believed to correspond to the outer region of the nucleolonema. Such convoluted formations disappear upon sufficiently long extraction with proteinase K. Using Bernhard's regressive staining technique for chromatin, the distribution of this substance throughout the nucleolar mass was found to match closely that of the nucleolonemata as revealed by several other procedures. As a last test for investigating the cytochemical make-up of the nucleolus, blocks of tissues were stained with 3,3'-diaminobenzidine, a substance known to react specifically with nucleic acids. When such specimens are digested with ribonuclease for 1 h, there persist within the nucleolus, fibrillogranular zones the localization of which is highly reminiscent of that of the nucleolonemata. Combination of ribonuclease hydrolysis with subsequent treatment with proteinase K (30 min) induces the extraction of a large proportion of the nucleolar material, the persisting loose and rather evenly distributed fibrils exhibiting a diamter of 3-5 nm. The possibility is considered that these units may correspond to chromatin fibrils although they have most likely been displaced from their original localization during the extraction procedures. Our cytochemical data suggest that, in Allium porrum, the nucleolonema is approximately 0.3 micrometer in diameter and may consist of a central axis from which chromatin loops project radially. A possible interpretation for the presence of protein-rich, 0.1 micrometer-thick, annular structures throughout the nucleolonemal skein is that the newly synthesized RNP products are accumulated transiently at the extremities of these loops before migrating to the immediately adjacent granular nucleolar zones.