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The Nucleolus Organizer and the Synaptonemal Complex in Endymion Non-Scriptus (L.)

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The Nucleolus Organizer and the Synaptonemal Complex in Endymion Non-Scriptus (L.) Jf. Cell Sci. 22, 75-86 (1976) 75 Printed in Great Britain THE NUCLEOLUS ORGANIZER AND THE SYNAPTONEMAL COMPLEX IN ENDYMION NON-SCRIPTUS (L.) E. G. JORDAN AND B. T. LUCK Biology Department, Queen Elizabeth College, University of London, Campden Hill Road, London W8, England SUMMARY Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The nucleolus went through the process of segregation at the beginning of meiosis with the movement to its surface of a pale-staining region. This region was shown to be the same as that called the 'L zone' or lacunae of nucleoli. Its chromosomal nature was strongly suggested by the presence of the synaptonemal complex within it. This demonstrated that the pale-staining region of nucleoli is the nucleolus organizer and almost certainly the chromosome region containing the ribosomal cistrons, and justifies the use of these terms to describe the structure when seen inside the nucleolus. The relationship between this zone and the hetero- chromatic knob called the nucleolar organizing body in maize by other workers is discussed. INTRODUCTION It has previously been reported from this laboratory that upon activation of cells from dormant Helianthus tuberosus tuber tissue, the lightly staining zone of the nucleolus, the 'L' zone, thought to be the nucleolus organizer, moves from an external to an internal position becoming dispersed as small threads within the fibrillar zone of the nucleolus (Jordan & Chapman, 1971). Nucleoli in cells not active in rRNA synthesis typically have discrete areas of fibrillar material and granular material, a condition which has been called the 'segregated' nucleolus (Bernhard, Frayssinet, Lafarge & Le Breton, 1965). Naturally segregated nucleoli have been reported in meiocytes of Allium cepa (Gimenez-Martin & Stockert, 1970), and recently Esponda & Gimenez-Martin (1975) have shown that the nucleolus organizer may be of variable form. The changes which occur between interphase and meiotic prophase should enable us to follow the processes which lead to the segregated appearance of inactive nucleoli and to decide the interphase counterpart of the nucleolus organizer which is so clearly apparent at meiosis. The evidence connecting the lightly staining zone of plant nucleoli in interphase with the nucleolus organizer is still indirect, resting mainly on its similarity in appearance to the nucleolus organizer identified in dividing cells, especially in lower plants (Godward & Jordan, 1965). It is well established that the rRNA genes reside in the secondary constriction of chromosomes (Ritossa & Spiegelman, 1965; Birnsteil, Wallace, Sirlin & Fishberg, 1966; Scheuermann & Knalmann, 1975), but the evidence that these secondary 76 E. G. Jordan and B. T. Luck constrictions are to be identified with the lightly staining zones or lacunae of plant nucleoli is not unequivocal. La Cour & Wells (1975) have expressed doubts about the organizer nature of the lightly staining regions preferring to restrict this term to the darker regions that have sometimes been reported within it. This paper reports the movement of the lightly staining zone from a position within the nucleolus during the interphase to the surface during late prophase of meiosis, where it would perhaps be recognized as the nucleolus organizer. This movement may be accompanied by a decrease in nucleolar RNA synthesis (Das, 1965; Das & Alfert, 1966; Parchman & Lin, 1972), and ribosome population of meiocytes (Mackenzie, Heslop-Harrison & Dickinson, 1967). The presence of the synaptonemal complex in the lightly staining zone is reported in this study and confirms its identification as a chromosomal region. Gillies (1973) has identified a somewhat similar structure as chromosomal in maize in the same way, but there a large knob of heterochromatin is found adjacent to it, which is also thought to be part of the nucleolus organizer. MATERIALS AND METHODS Endymion non-scriptus (L.) (Bluebell) plants were harvested in January. The cytological stage of the anthers in a floret was discovered by an aceto-orcein squash of one anther, the remaining 5 being sliced into 2-5 % distilled glutaraldehyde (Gillett & Gull, 1972), in 01 M phosphate buffer, pH 68 at room temperature. The material was left in glutaraldehyde for 4-5 h, thoroughly rinsed in buffer, postfixed for 2-3 h at room temperature in 1 % osmium tetroxide in the same buffer, dehydrated through an ethanol-propylene oxide series and embedded in Araldite. For electron microscopy, silver sections were cut with an LKB III ultramicrotome, stained with aqueous lead citrate (Reynolds, 1963), and post-stained in 10% (w/v) uranyl acetate in methanol (Stempack & Ward, 1964). Sections were examined with an AEI EM 6B electron microscope. For abbreviations used on figures, see p. 84. RESULTS Interphase nucleoli showed a peripheral granular region surrounding a fibrillar region; which contained lightly staining regions (Fig. 1) previously called the lightly staining or 'L' zone in other plant nucleoli (Jordan & Chapman, 1971, 1973)- Figs. 1-3. Meiocytes of Endymion non-scriptus (L.) showing rearrangement of the nucleolus during meiotic prophase. x 10000. Fig. 1. Interphase nucleus showing a nucleolus with an inner lightly staining zone. Fig. 2. Pre-leptotene nucleus showing the lightly staining zone towards the periphery of the nucleolus. A vacuole is present in the fibrillar region. Fig. 3. Pachetene-diplotene, large nucleolus completely segregated, with the lightly staining zone on the outside of the nucleolus. A synaptonemal complex can be seen in the heterochromatin. Fig. 4. Young microspore showing newly reformed nucleolus. Note no granular region and the production of accessory nucleoli from the lightly staining region. Exine is beginning to be formed at this time, x 10000. Nucleolus organizer and synaptonemal complex 77 Figs. 1-4. For legends see facing page. 78 E. G. Jordan and B. T. Luck In pre-leptotene nucleoli, the granular region enclosed the fibrillar region; however, the lightly staining zone by the onset of prophase had moved to a position just beneath the surface of the nucleolus. Connexions could be seen between the lightly staining zone and chromatin located outside the nucleolus (Fig. 2). By pachytene, the granular region had formed a cap on one side of the acentric fibrillar region. The lightly staining zone at this stage had assumed a position on the outside of the nucleolus adjacent to the acentric fibrillar region (Fig. 3). A similar position for the lightly staining zone was seen in newly reformed nucleoli at the tetrad stage (Fig. 4), but there, in the absence of a granular region. Accessory nucleoli were seen adjacent to the lightly staining zone and in the surrounding nucleoplasm. Following uranyl acetate and lead citrate staining, the synaptonemal complex was seen as a ribbon-like structure in intimate association with synapsed chromosomes (Figs. 5-7), having 2 dense amorphous lateral elements, and one central component surrounded by a less electron-dense space. In a lateral view the central component could be seen either as 2 parallel lines (Figs. 6, 13, 15), or as a single element (Fig. 7). Filaments were seen to traverse from the lateral element to the central element (Figs. 6, 7, 13-15). In transverse section these filaments were seen to occur in several planes and sometimes overlapping at the central element, where they appeared either to terminate (Figs. 8-10), or to extend to the opposite lateral element (Fig. 11). The dimensions found for the various components of the synaptonemal complex were as follows: central region width, 115 nm; lateral component diameter, 42-5 nm; central component diameter, 30 nm; transverse filament diameter, 7-5 nm. These dimensions accord with those for other monocotyledons (Westergaard & Von Wettstein, 1972). The figures above were based on a few measurements taken from the best sections. Sections of pachytene nucleoli showed the lightly staining zone of the nucleolus traversed by the undifferentiated synaptonemal complex whilst still partly embedded in the fibrillar region, yet at the same time clearly forming part of a chromosome (Figs. 12-15). The lightly staining zone which we designate the nucleolar organizer region at this time, is observed to be a region of irregular fibrils 5-12-5 nm in diameter, less intensely stained than chromatin. Figs. 5-11. Synaptonemal complexes in bivalents from zygotene nuclei of bluebells. Fig. 5. Frontal view showing synapsis of chromomeres along the length of the homologues. x 10500. Fig. 6. Frontal view showing fine structure of synaptonemal complex, x 30000. Fig. 7. Frontal radial section showing fine structure of synaptonemal complex, x 48750. Figs. 8—11. Transverse sections of synapsed chromosomes, showing the position of the fibrils which radiate from the lateral component towards the centre, x 48750. mmmmmozNucleolus organizer and synaptonemal complex 79 Figs. S~i i. For legends see facing page. 80 E. G. Jordan and B. T. Luck DISCUSSION The rearrangements of the nucleolus at meiosis, especially the relationships of the lightly staining zone of the nucleolus and the presence within it of the synaptonemal complex help to clarify our understanding of the nucleolus organizer and especially how it relates to the interphase nucleolus. Although in certain cell types the pale-staining regions of nucleoli have been identified as the nucleolus organizer, in others their nature is less certain. These lightly staining zones are commonly called
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