THE NUCLEOLUS ORGANIZER AND THE SYNAPTONEMAL COMPLEX...

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 electronmicroscopy. The nucleolus went through the process of segregation at the beginning of meiosiswith the movement to its surface of a pale-staining region. This region was shown to be thesame as that called the ' L zone' or lacunae of nucleoli. Its chromosomal nature was stronglysuggested by the presence of the synaptonemal complex within it. This demonstrated that thepale-staining region of nucleoli is the nucleolus organizer and almost certainly the chromosomeregion containing the ribosomal cistrons, and justifies the use of these terms to describe thestructure 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 cellsfrom dormant Helianthus tuberosus tuber tissue, the lightly staining zone of thenucleolus, the ' L ' zone, thought to be the nucleolus organizer, moves from anexternal to an internal position becoming dispersed as small threads within the fibrillarzone of the nucleolus (Jordan & Chapman, 1971). Nucleoli in cells not active in rRNAsynthesis typically have discrete areas of fibrillar material and granular material, acondition which has been called the 'segregated' nucleolus (Bernhard, Frayssinet,Lafarge & Le Breton, 1965). Naturally segregated nucleoli have been reported inmeiocytes of Allium cepa (Gimenez-Martin & Stockert, 1970), and recently Esponda& Gimenez-Martin (1975) have shown that the nucleolus organizer may be of variableform.

The changes which occur between interphase and meiotic prophase should enableus to follow the processes which lead to the segregated appearance of inactive nucleoliand to decide the interphase counterpart of the nucleolus organizer which is so clearlyapparent at meiosis.

The evidence connecting the lightly staining zone of plant nucleoli in interphasewith the nucleolus organizer is still indirect, resting mainly on its similarity inappearance to the nucleolus organizer identified in dividing cells, especially in lowerplants (Godward & Jordan, 1965).

It is well established that the rRNA genes reside in the secondary constriction ofchromosomes (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 plantnucleoli is not unequivocal. La Cour & Wells (1975) have expressed doubts aboutthe organizer nature of the lightly staining regions preferring to restrict this term tothe darker regions that have sometimes been reported within it.

This paper reports the movement of the lightly staining zone from a positionwithin the nucleolus during the interphase to the surface during late prophase ofmeiosis, where it would perhaps be recognized as the nucleolus organizer. Thismovement may be accompanied by a decrease in nucleolar RNA synthesis (Das,1965; Das & Alfert, 1966; Parchman & Lin, 1972), and ribosome population ofmeiocytes (Mackenzie, Heslop-Harrison & Dickinson, 1967).

The presence of the synaptonemal complex in the lightly staining zone is reportedin 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 thoughtto 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 0 1 M phosphate buffer, pH 6 8 at room temperature. The material was left inglutaraldehyde for 4-5 h, thoroughly rinsed in buffer, postfixed for 2-3 h at room temperaturein 1 % osmium tetroxide in the same buffer, dehydrated through an ethanol-propylene oxideseries and embedded in Araldite.

For electron microscopy, silver sections were cut with an LKB III ultramicrotome, stainedwith aqueous lead citrate (Reynolds, 1963), and post-stained in 10% (w/v) uranyl acetate inmethanol (Stempack & Ward, 1964). Sections were examined with an AEI EM 6B electronmicroscope. For abbreviations used on figures, see p. 84.

RESULTS

Interphase nucleoli showed a peripheral granular region surrounding a fibrillarregion; which contained lightly staining regions (Fig. 1) previously called the lightlystaining or ' L ' zone in other plant nucleoli (Jordan & Chapman, 1971, 1973)-

Figs. 1-3. Meiocytes of Endymion non-scriptus (L.) showing rearrangement of thenucleolus during meiotic prophase. x 10000.

Fig. 1. Interphase nucleus showing a nucleolus with an inner lightly stainingzone.

Fig. 2. Pre-leptotene nucleus showing the lightly staining zone towards theperiphery of the nucleolus. A vacuole is present in the fibrillar region.

Fig. 3. Pachetene-diplotene, large nucleolus completely segregated, with the lightlystaining zone on the outside of the nucleolus. A synaptonemal complex can be seenin the heterochromatin.

Fig. 4. Young microspore showing newly reformed nucleolus. Note no granularregion 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.


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 beneaththe surface of the nucleolus. Connexions could be seen between the lightly stainingzone and chromatin located outside the nucleolus (Fig. 2).

By pachytene, the granular region had formed a cap on one side of the acentricfibrillar region. The lightly staining zone at this stage had assumed a position on theoutside of the nucleolus adjacent to the acentric fibrillar region (Fig. 3).

A similar position for the lightly staining zone was seen in newly reformed nucleoliat the tetrad stage (Fig. 4), but there, in the absence of a granular region. Accessorynucleoli were seen adjacent to the lightly staining zone and in the surroundingnucleoplasm.

Following uranyl acetate and lead citrate staining, the synaptonemal complex wasseen as a ribbon-like structure in intimate association with synapsed chromosomes(Figs. 5-7), having 2 dense amorphous lateral elements, and one central componentsurrounded by a less electron-dense space. In a lateral view the central componentcould 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 severalplanes and sometimes overlapping at the central element, where they appeared eitherto terminate (Figs. 8-10), or to extend to the opposite lateral element (Fig. 11). Thedimensions found for the various components of the synaptonemal complex were asfollows: central region width, 115 nm; lateral component diameter, 42-5 nm; centralcomponent diameter, 30 nm; transverse filament diameter, 7-5 nm. These dimensionsaccord 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 nucleolustraversed by the undifferentiated synaptonemal complex whilst still partly embeddedin 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 organizerregion 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.


mmmmmoz

Figs. S~i i. For legends see facing page.


DISCUSSION

The rearrangements of the nucleolus at meiosis, especially the relationships of thelightly staining zone of the nucleolus and the presence within it of the synaptonemalcomplex help to clarify our understanding of the nucleolus organizer and especiallyhow it relates to the interphase nucleolus.

Although in certain cell types the pale-staining regions of nucleoli have beenidentified as the nucleolus organizer, in others their nature is less certain. Theselightly staining zones are commonly called lacunae but are quite different fromnucleolar vacuoles which have an appearance indistinguishable from nucleoplasm. Inmost cases where such lightly staining zones can be seen in nucleoli they show upbecause of their lower electron density in relation to both the chromatin and theother nucleolar areas (Jordan & Chapman, 1971, 1973; Lafontaine & Lord, 1973,1974), but in some situations the chromatin is indistinguishable from the lightlystaining zones (Godward & Jordan, 1965; Lafontaine, 1968; Jordan & Godward, 1969).In the latter situations it is not difficult to draw the conclusion that the lightly stainingzone in the nucleolus really is chromatin on the basis that it is both continuous withit and of identical appearance. But in those cells where there is a clear difference infibril size and stainability from the rest of the chromatin identification becomesindefinite. This is the case for Endymion non-scriptus.

Lightly staining zones have been recorded in animal cell nucleoli where they havebeen called 'fibrillar centres' and may or may not be the same structures, but thefact that in some cases they become apparent on the induction of nucleolar segregationpoints to their similarity (Recher, Parry, Briggs & Whitescarver, 1971; Recher,Briggs & Parr}', 1971).

The demonstration of the presence of DNA in such regions would provide evidencethat they were chromosomal. Some electron-microscope autoradiographic workprovides such evidence though a thorough statistical analysis of the result is notpresented (Lafontaine & Lord, 1973). Darkly staining chromatin present inside thezone has been shown to contain DNA by enzyme cytochemistry. However, this testdid 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 denselypacked, but slightly lighter-staining region, than the rest of the chromosome.Nuclear envelope breakdown is evident, x 12500.

t igs. 13, 14. Serial sections showing distinct boundaries between the nucleolusorganizer region and heterochromatin with traversing synaptonemal complex,x 18750.

Fig. 15. Higher resolution of the nucleolus organizer region. The chromatin ofthe NOR is seen to be composed of irregular fibrils 5-12-5 nm in diameter. Thedarkly staining structures appearing in zones of separation between the nucleolus andthe organizer are of unknown significance, x 25000.


:*: -^ "r^l^ff^-^a^s^^Sa®'

Figs. 12-15. For legends see facing page.


It is interesting that at meiosis in Endymion non-scriptus the lightly staining zoneof the nucleolus changes its position with the reorganization of nucleolar activity.As the nucleolus enlarges and segregates, this zone comes to the surface. All thesechanges can be understood in relation to the cessation of nucleolar activity. Thechange in position of this nucleolar zone is an exact reversal of the stages seen duringactivation 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), theirequivalence with the lightly staining zones of interphase nucleoli is strongly supportedby the rearrangements reported here in Endymion non-scriptus. Further support forthe use of the term nucleolus organizer is found at the meiotic stage in Endymionnon-scriptus because: the nucleolus adheres to it at prophase (Figs. 3, 12-15) and it isclearly part of the chromosome, as evidenced from the presence of the synaptonemalcomplex; nucleolar reorganization also occurs there at the tetrad stage, Fig. 4, withthe appearance of micronucleoli, which may be involved in the further ribosomere-population at this time (Williams et al. 1973).

Whether the term nucleolus organizer should be used for the lightly staining zonecan be discussed in relation to the nucleolar-organizing body reported in maizemeiocytes by McClintock (1934).

The structure called the nucleolar-organizing body by McClintock is a largeheterochromatic knob and not the pale-staining part of the chromosome. It is notthe part of the chromosome which forms secondary constrictions and which is nowknown to contain the rRNA cistrons. In McClintock's study of the 'organizer'translocation, the capacity for forming at least a small secondary constriction remainedwith both parts of the broken heterochromatic knob. The possibility exists that thisheterochromatin itself may not have been involved in the nucleolar reorganizationand may not merit the term nucleolar organizer body. The argument being that inboth cases 'nucleolar organizing' is performed by that chromatin which can formsecondary constrictions and not by the heterochromatic knob itself. When a break,apparently in the heterochromatic knob, occurs it cannot be concluded that nocistrons for nucleolar reorganization are found with the larger piece; on the contrarythe capacity of this part to make a nucleolus argues for their presence. It is notdifficult to envisage ways in which a part of the ribosomal cistron-containing segmentof the chromosome, which has the capacity to form secondary constrictions, mightbecome located in different regions of a chromosome. Perhaps some ribosomalcistrons had previously become located in the heterochromatin by an inversion, theoccurrence 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 largeheterochromatic knob in maize (Gillies, 1973), must not be taken to mean that anyribosomal cistrons are located in it and may be misleading. Gillies who refers to boththe heterochromatin knob and the lightly staining zone as organizer has reported thepresence of the synaptonemal complex in both. The lightly staining zone in maizecontains some denser material in addition and may not be in quite the same state aslightly staining zones without such darker regions.


The denser material within the lightly staining zone has been reported in otherplants by La Cour & Wells (1975) and they suggest that the rest of the structure, thepale-staining part, may not even be part of the organizer. However although densechromatin regions have often been reported in the lightly staining zone this situationdoes not always occur.

In Endymion non-scriptus there is no confusion because a counterpart of the largebody 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 ofHeitz & Bauer (1933), that the nucleolar organizer should really be equated with thesecondary constriction. But yet in maize we have a structure outside the secondaryconstriction 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 nowconclusive (Scheuermann & Knalmann, 1975), but whether the lightly staining zonesof 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 nucleolushas been provided by the demonstration of transcription at the electron-microscopelevel by Miller & Beatty (1969). This work has now been extended to plants by anumber 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 theproteins involved in the processing events it is not to be expected that they would bevisible in non-spread, conventional thin-section preparations. However, where anynumber of the rRNA cistrons take up a condensed or inactive configuration it seemsthat they appear with the characteristics of the lightly staining zone of nucleoli.Electron micrographs of secondary constrictions show the same pale-stainingcharacteristics as the lightly staining nucleolar zone (Lafontaine, 1968; Lafontaine &Lord, 1974). The secondary constrictions of animal cells have sometimes been shownto have a similarly lightly stained appearance (Hsu, Brinkley & Arrighi, 1967).

However the appearance of the ribosomal cistron-containing region of a chromosomeas a secondary constriction at mitosis is perhaps a function of the degree of con-densation of the chromatin, because in cells where there is no restriction on chromatincondensation, probably through lack of nucleolar activity, no region correspondingto the secondary constriction can be seen (McClintock, 1934; Hsu et al. 1967;Tanako & Terasaka, 1972). It is possible therefore that the secondary constrictionmay be different from other chromatin purely by this nucleolar hindrance tochromosome condensation in its vicinity. But the difference between the lightlystaining region of nucleoli from condensed chromatin at interphase, when theproteins 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 someorganisms show dense material within the lightly staining structure the term organizer

6-2


should not be restricted to the condensed or more darkly staining part. The lightlystaining zone may be the only structure present and from its connexion to thechromosome, position within the nucleolus and the presence of the synaptonemalcomplex, must be considered to be the organizer. The fact that the lightly stainingzone may condense into darkly staining material identical to condensed chromosomesneed 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 appearancethough when they do it can be argued that it is the result of incomplete condensationof chromatin and possibly a protein deficit in comparison with the rest of thechromosome.

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 zonein interphase.

The nucleolar organizer could then be considered as a chromatin region with atendency for incomplete condensation giving rise to the lightly staining zone andshowing different extents of complete condensation giving rise to different proportionsof darkly staining condensed chromatin regions. Nucleolar organizers may showvarying proportions of the 2 zones and even appear to be composed entirely of oneform or the other.

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

ABBREVIATIONS ON FIGURES

a accessory nucleoli (micronucleoli) g granular zone/ ' L ' region (nucleolus organizer)Ic lateral componentne nuclear envelopenuo nucleolussc synaptonemal complextf transverse filamentv vacuole

The material in all electron micrographs was fixed in 2-5 % distilled glutaraldehydebuffered at pH 6-8, postfixed in 1 % buffered osmium tetroxide and embedded inAraldite. Sections were stained in aqueous lead citrate followed by methanolicuranyl acetate.

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{Received 11 February 1976)

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