The Nucleolus and Meiosis During Microsporogenesis in Endymion Non-Scriptus (L.)

J. Cell Sci. 25, 111-123 (1977) 111 Printed in Great Britain

THE NUCLEOLUS AND MEIOSIS DURING MICROSPOROGENESIS IN ENDYMION NON-SCRIPTUS (L.)

B. T. LUCK AND E. G. JORDAN Biology Department, Queen Elizabeth College, University of London, Campden Hill Road, London WS 7 AH, England

SUMMARY Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The segregated components of the nucleolus at meiotic prophase underwent fragmentation and dissolution at pachytene-diplotene. Nucleoli were absent during both meiotic divisions and reformed on the nucleolus organizer into a fibrillar mass from scattered fibrillar components at the dyad and tetrad stages. It is argued that the fibrillar region shows continuity through nuclear division though undergoing structural transformations in the process. Nucleolar reformation occurs on condensed nucleolus organizers. Processing of the ribosomal precursors and the resumption of RNA synthesis is discussed in relation to the dispersal of the nucleolus organizer into the fibrillar region of the reformed nucleolus.

INTRODUCTION Nucleolar ultrastructure during the mitotic cycle has been extensively studied by various investigators (Lafontaine & Chouinard, 1963; Jordan & Godward, 1969; Pickett-Heaps, 1970; Noel, Dewey, Abel & Thompson, 1971; Chouinard, 1966, 1971, 1975; Lafontaine, 1958; Lafontaine & Lord, 1974). It is generally agreed that the nucleolus suffers a general dissolution during late prophase of mitosis in higher plants; however, there are differing reports on the mode of its dissolution and the subsequent fate of the different parts (Lafontaine & Lord, 1969; Chouinard, 1971; Moreno Diaz De La Espina, Risueno, Fernandez-Gomez & Tandler, 1976). The origin of the material which constitutes the reappearing nucleoli is also still a matter of considerable uncertainty. Cytochemical studies of Allium cepa meristematic cells (Stockert, Fernandez-Gomez & Gimenez-Martin, 1970; Gimenez-Martin, De La Torre, Fernandez-Gomez & Gonzalez-Fernandez, 1974; Moreno Diaz De La Espina et al. 1976) support the earlier view that prenucleolar bodies, seen in the newly formed nucleus, arise from the so called pre-nucleolar material seen around the anaphase chromosomes (McClintock, 1934; Lafontaine, 1958). However, the contri- bution to the new nucleoli of the so called pre-nucleolar fibrillo-granular material found around the anaphase chromosomes is questioned by some workers (Swift, 1959; Chouinard, 1966; Lafontaine & Lord, 1974). The extent to which the new nucleolus is dependent on de novo synthesis has been followed in recent experiments employing inhibitors of RNA and protein synthesis (Gimenez-Martin et al. 1974; Semeshin, Sherudilo & Belyaeva, 1975; Risueno, Moreno Diaz De La Espina, Fernandez- Gomez & Gimenez-Martin, 1976). ii2 B.T. Luck and E. G. Jordan The synchrony of the meiotic process and the greater duration especially of the phase of nucleolar breakdown in the extended prophase of meiosis, together with the fact that there are 2 phases of reorganization, one after each division, only one of which leads to a normal interphase, makes it a useful and interesting system for studies on the nucleolar cycle. As previously reported (Jordan & Luck, 1976), concomitant with nucleolus segregation during meiotic prophase in Endymion non-scrtptus, the nucleolus organizer emerges to the surface of the nucleolus. In this paper we report the various rearrange- ments of the nucleolar zones in the prophase breakdown and the subsequent reorganization events after each of the 2 meiotic divisions. This work indicates that some of the constituents of the new nucleolus are synthesized before nucleolar reorganization. Thus the understanding of the nucleolus becomes not only the understanding of the synthesis of nucleolar materials but also their dispersal and reorganization at nuclear division.

MATERIALS AND METHODS Endymion non-scriptus (L.) (Bluebell) plants were harvested in mid-January. The cyto- logical stage of the anthers in a floret was found from an aceto-orcein squash of one anther. The remaining 5 were sliced into 3% distilled glutaraldehyde in 01 M phosphate buffer, pH 6-8 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. Sections were cut using a diamond knife, stained with aqueous lead citrate and post-stained with 10% (w/v) uranyl acetate in methanol (Stempack & Ward, 1964), and examined in an AEI EM 6B electron microscope.

Labelling on figures a accessory nucleoli U lipid droplet c chromatin in mitochondrion cli chromosome nd nucleoid ci cytoplasmic imagination ne nuclear envelope f fibrillar region np nuclear pore ff fibrillar region fragment pb pre-nucleolar body g granular region psc polysynaptonemal complex if granular region fragment me remnant nuclear envelope go Golgi sc synaptonemal complex 1 lightly staining region V vacuole

Figs. 1-3. Electron micrographs showing nucleolus segregation and fragmentation. Fig. 1. Segregated zygotene nucleolus with the nucleolus organizer in an external position. An accessory nucleolus is on the surface of the nucleolus organizer and cytoplasmic invaginations are present in the nucleus, x 12500. Fig. 2. Pachytenc-diplotene. High magnification of nucleolus organizing region showing fibrils of 5-125 nm in diameter. The accessory nucleoli have a similar fibril size to the fibrillar region: 5-10 nm, and the fibrils of the darkly staining chromatin are 10-15 nm m diameter, x 32000. Fig. 3. Pachytene-diplotene. There is a clear separation of the various parts of the nucleolus; the fibrillar region has fragmented. An accessory nucleolus and a remaining synaptonemal complex are seen in the diffuse chromatin. Few nuclear pores are observed in the nuclear envelope, x 11000. The nucleolus during microsporogenesis 113 II4 B. T. Luck and E. G. Jordan The nucleolus during microsporogenesis 115

RESULTS Nucleolar segregation, separation and dissolution Zygotene nucleoli displayed the typical 'segregated nucleolus' appearance and also had some accessory nucleoli associated with the nucleolus organizer (Fig. 1). Accessory nucleoli had a similar staining intensity to the fibrillar region (Fig. 1), with fibrils between 5 and 10 nm in diameter. The chromatin fibrils were 10-15 nm m diameter (Fig. 2). By diplotene, the various components had separated (Fig. 3). These separate parts of the nucleolus also showed evidence of fragmentation (Figs. 3-6). During the final stages of nucleolar dissolution when the fibrillar and granular zones had moved apart from each other and were breaking up there was no indication of the fate of the granules or fibrils. The small areas of separate fibrillar and granular zones simply became smaller until they eventually disappeared. From the compactness of the fibrillar pieces during this process it seems that fibrils must be leaving the surface to reduce the size of the fragments, rather than gradually dispersing en masse by a general disaggregation. The granular region, however, although staying in recognizable pieces, does seem to disperse by becoming generally looser until the granules cannot be distinguished from the background. At this time there is an increase in the number of granules seen in the nucleoplasm. It is difficult to decide whether or not any of the nucleolar materials are contributing to the matrix of the recondensing chromosomes because they are still very dispersed at this time. During nucleolar dissolution large areas of the nuclear envelope remain intact (Figs. 3, 4). Frequently, large pieces of nucleolar granular zone were seen in apposition to the nuclear envelope (Fig. 6).

Chromosomes at metaphase 1 and nucleolar reformation at the dyad stage Meiotic metaphase chromosomes showed a homogenous fibrous appearance (Fig. 7) with individual fibrils between 6 and 15 nm in diameter (Fig. 8). During chromosome decondensation at the time of the newly formed dyad, numerous fibrous bodies were seen adjacent to the chromosomes and free in the nucleoplasm (Fig. 9). As decondensation progressed these bodies decreased in number and increased in size (Figs. 10-12), and were seen to be composed of fibrils 5-10 nm in diameter (Fig. 12). These pre-nucleolar bodies coalesced on to the nucleolus organizer forming the main nucleolus which was composed of a fibrillar mass with a lightly staining zone on the surface (Figs. 13, 14), essentially similar to the inactive nucleoli described in Helianthus tuberosus (Jordan & Chapman, 1971).

Figs. 4-6. Various aspects of nucleolar dissolution. Figs. 4, 5. Pachytene-diplotene, showing the fragmentation of the granular and fibrillar region of the nucleolus. Fig. 4, x 16500; Fig. 5, x 9500. Fig. 6. Diplotene. A large granular region fragment is seen in apposition to the remnant nuclear envelope, x 12750. n6 B. T. Luck and E. G. Jordan

m The nucleohis during microsporagenesis 117

Chromosomes at anaphase 2 and nucleolar reformation at the tetrad stage Anaphase 2 chromosomes showed loosely arranged fibrils 6-15 nm in diameter (Fig. 15). Nucleolar reformation at the tetrad stage occurred in a similar fashion to that at the dyad stage, with the appearance of pre-nucleolar bodies which coalesced on to the nucleolus organizer (Fig. 16) to form the main nucleolus of the young tetrad, which was also composed of a large fibrillar mass with a lightly staining zone on the surface (Fig. 17). When primary exine development was complete the young microspores were seen to have nuclei with a few areas of condensed chromatin surrounded by large areas of dispersed chromatin. The lightly staining zone of the nucleolus was then seen in the interior of the fibrillar region. Concurrent with this rearrangement was the appearance of a peripheral granular region, and the production of some nuclear bodies (Fig. 18). Such nucleoli had the same structural characteristics as those of artichoke nuclei with activated ribosomal cistrons (Jordan & Chapman, 1971).

DISCUSSION During meiotic prophase in Endymion non-scriptus the nucleoli undergo segregation of their different zones with the appearance of the organizer as a discrete mass on the surface of the fibrillar region (Jordan & Luck, 1976). Two separate nucleoli will be brought together by the synapsing of their organizers. This explains how only a single large nucleolus is seen at pachytene. The accessory nucleoli seen at this stage have an ultrastructure similar to the fibrillar zone, sometimes denser, and can be readily distinguished from the chromatin by the diameter of their constituent fibrils. They are not thought to be a condensed part of the nucleolus organizer (La Cour & Wells, 1975). Although the nucleolus organizer may eventually condense and appear as darkly stained as the rest of the chromatin, the time when this happens may vary with species. In Endymion non- scriptus the organizer is pale-staining at pachytene, a condition also seen in Lilium longiflorum (Williams, Heslop-Harrison & Dickinson, 1973), but in Allium cepa the nucleolus organizer is only partially lightly stained (Esponda & Gimenez-Martin,

Figs. 7—10. Metaphase 1 chromosomes and the appearance of pre-nucleolar bodies during chromatin decondensation. Fig. 7. Metaphase 1 chromosomes show a homogeneous fibrillar appearance. The broken down nuclear envelope forms a discontinuous membrane around the spindle area. A few cytoplasmic organelles are seen in the spindle area, x 5100. Fig. 8. Higher magnification of a metaphase 1 chromosome which shows it to be composed of fibrils between 6 and 15 nm in diameter, x 31000. Fig. 9. Newly formed dyad. Prenucleolar bodies are adjacent to the decondensing chromosomes and free in the nucleoplasm. A nucleoloid is present in the cytoplasm. Nuclear pores (arrowheads), x 10000. Fig. 10. A later stage of the dyad as judged by the further decondensation of the chromosomes and the larger pre-nucleolar bodies. A polysynaptonemal complex is present in the nucleoplasm. x 10000. n8 B. T. Luck and E. G. Jordan The nucleolus during microsporogenesis 119 1975). We have not seen lightly staining zones in stages later than pachytene-diplotene until the times of nucleolar reformation after both divisions, when the nucleolus organizer has the characteristic lightly stained appearance. During diplotene desynapsis the separated fibrillar and granular zones of the nucleolus become fragmented. The chromosomes become very diffuse at pachytene- diplotene, and the remaining fragments of the nucleolus disappear. It is difficult to decide upon the fate of the dispersed regions of the nucleolus. However, it is con- ceivable that some ribosomal precursor fibrils will become part of the matrix of condensing chromosomes (Lafontaine & Chouinard, 1963; Lafontaine, 1958). This matrix can be considered to be arrested nucleolar machinery, in particular its high molecular weight ribosomal RNA precursors associated with the metaphase chromosomes (Fan & Penman, 1971). The consequence of this would be that some nucleolar formation could occur without any requirement for concurrent synthesis. Although some RNA may be synthesized during telophase in mitotic divisions (Prescott & Bender, 1962; Monesi, 1964), evidence shows that pre-nucleolar bodies can be formed in the absence of RNA synthesis (Stevens & Prescott, 1971; Stockert et al. 1970; Phillips, 1972; Gimenez-Marti'n et al. 1974; Semeshin et al. 1975; Risueno et al. 1976) or protein synthesis (Stockert etal. 1970; Gimenez-Marti'n et al. 1974). Further, even under conditions of normal protein synthesis the reappearance of nucleoli at the end of mitosis involved the utilization of proteins which were synthesized before mitosis (Harris, 1961). There is thus good evidence that some of the remnants of the dispersed prophase nucleolus are reutilized in nucleologenesis. In anaphase and telophase of mitosis a fibrillo-granular material has been reported coating the chromosomes. Whether or not such a fibrillo-granular material is a stage in nucleolar reformation has been a matter of some debate. The material has even been called the pre-nucleolar material. But because such a material has clearly defined granules which do not appear in the first recognizable nucleolar precursor bodies it has been argued that it is not nucleolar precursor material (Chouinard, 1966, 1971). In this work on Endymion non-scriptus at meiosis no material having the precise nature of a fibrillo-granular coating of the chromosomes was seen. However, the areas between the decondensing chromosomes do show the presence of scattered granules which may be its counterpart, and it is possible that a stage showing a fibrillo-granular coating of the chromosomes occurs but is of short duration and has therefore not been

Figs. 11—14. Nucleologenesis at the dyad stage. Fig. 11. Later stage of the dyad than Figs. 9, io, as judged by chromatin dispersal, fewer and larger pre-nucleolar bodies, arrowheads, nuclear pores, x 10000. Fig. 12. Higher magnification of outlined rectangular area of Fig. 11, showing the pre-nucleolar bodies to be composed of fibrils 5-10 nm in diameter, x 39000. Fig. 13. Large fibrillar mass adjacent to 2 lightly staining nucleolus organizer regions which are continuous with more darkly staining condensed chromatin. A pre-nucleolar body is present in the nucleoplasm. x 17250. Fig. 14. Main nucleolus of the dyad composed of a large fibrillar region with a lightly staining organizer region embedded in its surface, x 14500. 120 fi. 7\ Luck and E. G. Jordan The nucleolus during microsporogenesis 121 observed. Our evidence would argue against any involvement of a granular material in nucleologenesis, the fibrillar pre-nucleolar bodies appearing in scattered places amongst the chromosomes in a manner consistent with an origin from some fibrillar chromosomal matrix. The appearance of a fibrillo-granular mass in mitosis might well be the expression of non-nucleolar RNA synthesis in such a scheme of gene reprogramming as proposed by Goldstein (1976) and may then be quite different for meiosis. Whether the pre- nucleolar bodies and nucleoli arise from a chromosomal fibrillar matrix directly (Lafontaine & Lord, 1974) or whether the so called pre-nucleolar material around anaphase chromosomes must be an intermediate cannot yet be settled, but a more detailed investigation of the 2 meiotic anaphases could help to clarify the situation. The collection of the pre-nucleolar bodies into one nucleolus, the process of nucleolar reorganization, has been shown to be dependent on the presence of a nucleolus organizer. In the absence of organizers the many pre-nucleolar bodies, though undergoing some enlargement and perhaps fusion, remain separate or 'un- organized' (Swift & Stevens, 1966). From the use of inhibitors it has been concluded that nucleolar organization is dependent on RNA synthesis (Risueno et al. 1976). Our observations show the effect of the organizer in marshalling the pre-nucleolar bodies to form the large fibrillar mass of the newly reformed nucleolus. The external condensed organizer of the newly reformed nucleolus has a strong resemblance to the structure of an inactive organizer (Jordan & Chapman, 1971, 1973), suggesting that nucleolar reorganization can be performed by an organizer that is not synthesizing rRNA. Nucleolar reformation in the absence of rRNA synthesis is in line with the actinomycin D experiments of Semeshin et al. 1975. Perhaps there is some force of attraction between the nucleolus organizer and nucleolar material which is not dependent on RNA synthesis. A further indication that a force of 'attraction' exists between the organizer and the nucleolar material even when the organizer is not active in synthesis is the fact that the fibrillar regions of nucleoli remain closely adherent to withdrawn condensed inactive organizers. However, the earlier dispersed location of the fibrillar pre-nucleolar bodies is difficult to explain if it is argued that even a synthetically inactive organizer could suffice for nucleolar reorganization, because such exists at the time when these structures first appear. Although nucleolar organization may not require synthesis or extensive decondensation of the organizer

Fig. 15. Anaphase 2 chromosome composed of loosely arranged fibrils 6-15 nm in diameter, x 17250. Fig. 16. Pre-nucleolar bodies of the young tetrad. A pre-nucleolar body has coalesced on to the lightly stained, condensed chromatin region (nucleolus organizer). x 17250. Fig. 17. Newly reformed nucleolus of the young tetrad composed of a large fibrillar mass with the nucleolus organizer on the surface. Nucleolar vacuoles are present within the fibrillar region, x 21 375. Fig. 18. Nucleolus of young microspore with the nucleolus organizer in the interior of the fibrillar region which is surrounded by granular regions. A spherical nuclear body is on the surface of the fibrillar region, x 19125. 122 B. T. Luck and E. G. Jordan chromatin, it might be argued that at least some chromatin from the apparently condensed organizer had begun to decondense in the reorganization process, even though most of it could still be recognized as a condensed structure, the lightly staining zone. Further work on the amount of RNA synthesis associated with condensed organizers especially at the end of anaphase I of meiosis could help to settle the question. The dispersion of the nucleolus organizer into the nucleolus is a process which accompanies the arrival of a granular zone (Jordan & Chapman, 1971, 1973)- The outcome of nucleologenesis at the 2 meiotic divisions is different in this respect. While at the dyad a nucleolus is organized at the condensed organizer, it remains a purely fibrillar structure not developing a granular zone, while at the tetrad stage the nucleolus organizer moves into or becomes engulfed by the enlarging nucleolus as the granular zone appears. If the granules are evidence of resumed nucleolar function then nucleologenesis in the first division occurs without any resumption of RNA synthesis or processing, while at the end of the second meiotic division nucleologenesis results in both the resumption of rRNA synthesis and processing. This is consistent with Taylor's (1958) interpretation of his experiments on the time of RNA synthesis in Tulbaghia. It is therefore possible to speculate that the nucleolus organizing region may collect a fibrillar zone while remaining largely in a condensed con- figuration, but that its dispersal must precede or accompany any restoration of RNA synthesis or processing.

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