Jf. 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 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 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 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 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 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 . 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 lacunae but are quite different from nucleolar which have an appearance indistinguishable from nucleoplasm. In most cases where such lightly staining zones can be seen in nucleoli they show up because of their lower electron density in relation to both the chromatin and the other nucleolar areas (Jordan & Chapman, 1971, 1973; Lafontaine & Lord, 1973, 1974), but in some situations the chromatin is indistinguishable from the lightly staining zones (Godward & Jordan, 1965; Lafontaine, 1968; Jordan & Godward, 1969). In the latter situations it is not difficult to draw the conclusion that the lightly staining zone in the nucleolus really is chromatin on the basis that it is both continuous with it and of identical appearance. But in those cells where there is a clear difference in fibril size and stainability from the rest of the chromatin identification becomes indefinite. This is the case for Endymion non-scriptus. Lightly staining zones have been recorded in animal cell nucleoli where they have been called 'fibrillar centres' and may or may not be the same structures, but the fact that in some cases they become apparent on the induction of nucleolar segregation points to their similarity (Recher, Parry, Briggs & Whitescarver, 1971; Recher, Briggs & Parr}', 1971). The demonstration of the presence of DNA in such regions would provide evidence that they were chromosomal. Some electron-microscope autoradiographic work provides such evidence though a thorough statistical analysis of the result is not presented (Lafontaine & Lord, 1973). Darkly staining chromatin present inside the zone has been shown to contain DNA by enzyme cytochemistry. However, this test did not indicate the presence of DNA in the surrounding more lightly staining region (Chouinard, 1970).

Figs. 12-15. Synapsed bivalents of 'L' zone (NOR). Fig. 12'. The lightly staining zone or nucleolus organizer region appears as a densely packed, but slightly lighter-staining region, than the rest of the chromosome. breakdown is evident, x 12500. tigs. 13, 14. Serial sections showing distinct boundaries between the nucleolus organizer region and heterochromatin with traversing synaptonemal complex, x 18750. Fig. 15. Higher resolution of the nucleolus organizer region. The chromatin of the NOR is seen to be composed of irregular fibrils 5-12-5 nm in diameter. The darkly staining structures appearing in zones of separation between the nucleolus and the organizer are of unknown significance, x 25000. Nucleolus organizer and synaptonemal complex 81

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Figs. 12-15. For legends see facing page. 82 E. G. Jordan and B. T. Luck It is interesting that at meiosis in Endymion non-scriptus the lightly staining zone of the nucleolus changes its position with the reorganization of nucleolar activity. As the nucleolus enlarges and segregates, this zone comes to the surface. All these changes can be understood in relation to the cessation of nucleolar activity. The change in position of this nucleolar zone is an exact reversal of the stages seen during activation of cells from dormant plant storage tissue (Jordan & Chapman, 1971, 1973)- Although the nucleolus organizers of meiocytes have been identified at the electron- microscope level before (Williams, Heslop-Harrison & Dickenson, 1973), their equivalence with the lightly staining zones of interphase nucleoli is strongly supported by the rearrangements reported here in Endymion non-scriptus. Further support for the use of the term nucleolus organizer is found at the meiotic stage in Endymion non-scriptus because: the nucleolus adheres to it at prophase (Figs. 3, 12-15) and it is clearly part of the chromosome, as evidenced from the presence of the synaptonemal complex; nucleolar reorganization also occurs there at the tetrad stage, Fig. 4, with the appearance of micronucleoli, which may be involved in the further ribosome re-population at this time (Williams et al. 1973). Whether the term nucleolus organizer should be used for the lightly staining zone can be discussed in relation to the nucleolar-organizing body reported in maize meiocytes by McClintock (1934). The structure called the nucleolar-organizing body by McClintock is a large heterochromatic knob and not the pale-staining part of the chromosome. It is not the part of the chromosome which forms secondary constrictions and which is now known to contain the rRNA cistrons. In McClintock's study of the 'organizer' translocation, the capacity for forming at least a small secondary constriction remained with both parts of the broken heterochromatic knob. The possibility exists that this heterochromatin itself may not have been involved in the nucleolar reorganization and may not merit the term nucleolar organizer body. The argument being that in both cases 'nucleolar organizing' is performed by that chromatin which can form secondary constrictions and not by the heterochromatic knob itself. When a break, apparently in the heterochromatic knob, occurs it cannot be concluded that no cistrons for nucleolar reorganization are found with the larger piece; on the contrary the capacity of this part to make a nucleolus argues for their presence. It is not difficult to envisage ways in which a part of the ribosomal cistron-containing segment of the chromosome, which has the capacity to form secondary constrictions, might become located in different regions of a chromosome. Perhaps some ribosomal cistrons had previously become located in the heterochromatin by an inversion, the occurrence of an inversion at the same time as the translocation cannot be auto- matically excluded. The continued use of the term nucleolar organizer for the large heterochromatic knob in maize (Gillies, 1973), must not be taken to mean that any ribosomal cistrons are located in it and may be misleading. Gillies who refers to both the heterochromatin knob and the lightly staining zone as organizer has reported the presence of the synaptonemal complex in both. The lightly staining zone in maize contains some denser material in addition and may not be in quite the same state as lightly staining zones without such darker regions. Nucleolus organizer and synaptonemal complex 83 The denser material within the lightly staining zone has been reported in other plants by La Cour & Wells (1975) and they suggest that the rest of the structure, the pale-staining part, may not even be part of the organizer. However although dense chromatin regions have often been reported in the lightly staining zone this situation does not always occur. In Endymion non-scriptus there is no confusion because a counterpart of the large body called the nucleolar organizing body does not appear and there are no darker- staining regions within the lightly staining zone. This confirms the earliest views of Heitz & Bauer (1933), that the nucleolar organizer should really be equated with the secondary constriction. But yet in maize we have a structure outside the secondary constriction being called the nucleolar organizing body and by some the 'Knor' (i.e. heterochromatic knob of the nucleolar organizer). Evidence that the secondary constrictions do contain the rRNA cistrons is now conclusive (Scheuermann & Knalmann, 1975), but whether the lightly staining zones of interphase nucleoli can be equated with them is still not clear (La Cour & Wells, 1967; Jordan & Chapman, 1971, 1973; Rose & Setterfield, 1971; Lafontaine & Lord, 1973, 1974; Chouinard, 1974). Conclusive evidence for the presence of DNA coding for rRNA in the nucleolus has been provided by the demonstration of transcription at the electron-microscope level by Miller & Beatty (1969). This work has now been extended to plants by a number of workers (Trendelenburg, Spring, Scheer & Franke, 1974; Spring, Trendelenburg, Scheer & Franke, 1974; Woodcock, Stanchfield & Gould, 1975). Since the genes are covered in the products of their synthesis together with the involved in the processing events it is not to be expected that they would be visible in non-spread, conventional thin-section preparations. However, where any number of the rRNA cistrons take up a condensed or inactive configuration it seems that they appear with the characteristics of the lightly staining zone of nucleoli. Electron micrographs of secondary constrictions show the same pale-staining characteristics as the lightly staining nucleolar zone (Lafontaine, 1968; Lafontaine & Lord, 1974). The secondary constrictions of animal cells have sometimes been shown to have a similarly lightly stained appearance (Hsu, Brinkley & Arrighi, 1967). However the appearance of the ribosomal cistron-containing region of a chromosome as a secondary constriction at is perhaps a function of the degree of con- densation of the chromatin, because in cells where there is no restriction on chromatin condensation, probably through lack of nucleolar activity, no region corresponding to the secondary constriction can be seen (McClintock, 1934; Hsu et al. 1967; Tanako & Terasaka, 1972). It is possible therefore that the secondary constriction may be different from other chromatin purely by this nucleolar hindrance to chromosome condensation in its vicinity. But the difference between the lightly staining region of nucleoli from condensed chromatin at interphase, when the proteins of mitotic condensation cannot be implicated (Huberman & Attardi, 1966; DuPraw & Bahr, 1969) must also be explained. In conclusion, it can be said that though the nucleolar organizer may in some organisms show dense material within the lightly staining structure the term organizer 6-2 84 E. G. Jordan and B. T. Luck should not be restricted to the condensed or more darkly staining part. The lightly staining zone may be the only structure present and from its connexion to the chromosome, position within the nucleolus and the presence of the synaptonemal complex, must be considered to be the organizer. The fact that the lightly staining zone may condense into darkly staining material identical to condensed chromosomes need not force us to the conclusion that some of it must always be in that state. Secondary constrictions do not always have the more lightly stained appearance though when they do it can be argued that it is the result of incomplete condensation of chromatin and possibly a deficit in comparison with the rest of the chromosome. It is not impossible that a difference in protein content and an incomplete con- densation is responsible for the appearance of the organizer as a lightly staining zone in interphase. The nucleolar organizer could then be considered as a chromatin region with a tendency for incomplete condensation giving rise to the lightly staining zone and showing different extents of complete condensation giving rise to different proportions of darkly staining condensed chromatin regions. Nucleolar organizers may show varying proportions of the 2 zones and even appear to be composed entirely of one form or the other.

We express our thanks to Dr K. R. Lewis for helpful discussion and for reading the manuscript and to Dr B. Bainbridge, Dr J. Chapman, Mr R. Gillett and Dr N. Severs for their useful advice. A grant from the Central Research Fund, University of London, made possible the purchase of a diamond knife.

ABBREVIATIONS ON FIGURES a accessory nucleoli (micronucleoli) g granular zone c chromatin / 'L' region (nucleolus organizer) dc diffuse chromatin Ic lateral component he heterochromatin ne nuclear envelope cc central component nuo nucleolus ch chromomere sc synaptonemal complex cr central region tf transverse filament e exine v vacuole f fibrillar zone The material in all electron micrographs was fixed in 2-5 % distilled glutaraldehyde buffered at pH 6-8, postfixed in 1 % buffered osmium tetroxide and embedded in Araldite. Sections were stained in aqueous lead citrate followed by methanolic uranyl acetate.

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{Received 11 February 1976)