an ultrastructural and radioautographic study …examination of semithin longitudinal section...

J. Cell Sci. 31, 193-207 (1976) 193

Printed in Great Britain

AN ULTRASTRUCTURAL AND

RADIOAUTOGRAPHIC STUDY OF THE

CHROMOCENTRIC INTERPHASE NUCLEUS IN

PLANT MERISTEMATIC CELLS (RAPHANUS

SATIVUS)

A. LORD AND J. G. LAFONTAINELaboratoire de Biologic cellulaire et moliculaire, Departement de Biologie,Faculty des Sciences et de G&rie, Universitd Laval, Quebec, Canada, GiK 7P4

SUMMARY

In Raphanus sativus, the mitotic chromosomes are quite short and, on reaching the cellpoles, soon undergo extensive unravelling. By late telophase and early interphase, only a fewchromosome segments, believed to correspond to the centromeric regions, are still visible inthe form of chromocentres closely associated with the nuclear envelope.

Although interphase nuclei show little internal structural differentiation, high-resolutionradioautography has permitted us to establish which of them have reached the early, mid and lateS periods. In early 5 nuclei, only the nucleolus and the euchromatin which pervades the nuclearcavity become labelled. By the mid S-period, the diffuse chromatin and nucleolus incorporateless thymidine and DNA synthesis is initiated within the peripheral chromocentres. Sub-sequently, the radioautographic grains become restricted to the chromocentres. The findingthat certain late S nuclei exhibit loosely organized chromocentres strongly suggests that theseheterochromatic chromosome segments undergo important conformational modifications dur-ing DNA replication. Finally, the presence of radioautographic grains over the lacunar regionsof the nucleolus in early and mid S nuclei demonstrates that intranucleolar DNA replicatesduring the earlier portion of the 5-period.

INTRODUCTION

Plant interphase nuclei are known from light-microscopical observations to differmarkedly in organization with species (reviewed in Dangeard, 1947; Delay, 1948;Lafontaine, 1968). Certain plants (e.g. Allium cepa, Vicia faba) are characterized bylarge DNA contents, correspondingly long mitotic chromosomes and highly struc-tured interphase nuclei. On account of the many advantages which these species pro-vide for studying the morphology of chromosomes during the cell cycle, they haveattracted much attention in the past (reviewed in Kaufmann, Gay & McDonald, i960;Ris, 1961; Moses, 1964; Lafontaine, 1974). Other plants possess much shorter mitoticchromosomes and, except for peripheral heterochromatic masses and the nucleolus,their interphase nuclei, known as chromocentric nuclei, appear rather homogeneousin organization. Contrary to reticulate plant nuclei, the ultrastructure of which hasrecently been the object of detailed observations (Kuroiwa & Tanaka, 1970, 1971;Kuroiwa, 1974; Lafontaine & Lord, 1974a), little information has appeared, so far,

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194 A. Lord and J. G. Lafontaine

on the ultrastructural organization of chromocentric interphase nuclei (Peveling,1961; Buvat, 1963).

The present study describes the gross and ultrastructural organization of the nu-cleus, from early telophase to late interphase, in a species (Raphanus sativus) of thechromocentric type. In view of the difficulty, in this species, of determining whatperiod of interphase a given nucleus has reached, material was labelled with tritiatedthymidine and S nuclei were identified by radioautography.

MATERIAL AND METHODS

Roots of Raphanus sativus grown in damp Vermiculite were used for the present investigation.For radioautography, roots were immersed in water containing from 5 to 20 /iCi/ml of [6-3H]-thymidine (sp. act.: 8 or 10 Ci/mmol) for periods varying from 15 to 60 min. This precursorwas obtained from Schwarz-Mann, Orangeburg, N.Y. For other experiments roots exposedto tritiated thymidine for 15 min were subsequently transferred to water for different periods,up to 4 h. Post-treatment with ' cold' thymidine was avoided in the course of these studies sincecontrol observations under electron microscopy have revealed that this DNA precursor tendsto perturb the organization of nuclei. Also, according to earlier findings, the reserve pool oftritiated thymidine in meristematic plant cells is very small and is used up within a few minutesfollowing transfer of the roots to water (Evans, 1964). All the roots were fixed either in 4 %glutaraldehyde or in 1 % osmium tetroxide adjusted to pH 7-2 with cacodylate buffer. Theywere then dehydrated in acetone and embedded in glycol methacrylate or in Epon 812 accordingto current procedures.

For light microscopy, semi-thin sections (0-5-1-0 flm) were deposited on glass slides andstained either with 1 % methylene blue in a 1 % aqueous sodium borate solution or by theFeulgen procedure. Some sections were also placed on quartz slides for ultraviolet observa-tions with a Zeiss UMSP-i instrument.

Light-microscope radioautography was performed on oy-fim Epon sections affixed to glassslides and coated by dipping in a Kodak NTB-2 bulk emulsion. Following appropriate ex-posure, the preparations were developed in Kodak Dektol and stained with methylene blue. Forhigh-resolution radioautography, ultrathin sections were first deposited on collodion-coatedslides and then covered with Ilford L-4 bulk emulsion diluted in 3 volumes of water. Following1-2 month exposure, the preparations were developed by the gold latensification-Elon ascorbicacid method according to Wisse & Tates (1968). The ultrathin sections were then transferredto copper grids, stained with both uranyl acetate and lead citrate, and examined in a PhilipsEM 300 electron microscope provided with an anti-contamination device.

Fig. 1. Micrograph of anaphase chromosomes (Raphanus sativus). The latter are onlya couple of microns in length and show little recognizable structural detail. Phase-contrast picture of o-7-/tm section stained by the Feulgen procedure and counterstainedwith 1 % methylene blue, x 6000.

Fig. 2. Micrograph of 2 daughter early to mid-telophase nuclei. The chromosomes areimmersed in a pervading material, the socalled prenucleolar substance, and theirstructure is not easily analysed. It is evident, however, that they have already startedunravelling with the result that only certain segments remain clearly perceptible.Phase-contrast micrograph of o-y-jim section stained as in Fig. 1. x 6000.

Fig. 3. In these nuclei the chromosomes have largely unravelled and only small denseportions remain in the form of chromocentres closely associated with the nuclearenvelope and immersed within patches of prenucleolar substance. This lattermaterial has partly disappeared and large transparent nucleoplasmic zones have be-come visible. In view of its irregular contours and the presence of emerging nucleoli(arrow), this nucleus is taken to belong to the early G]-period (Lafontaine & Lord,19746). Phase-contrast micrograph of specimen stained as in Fig. 1. x 6000.

Ultrastructure of chromocentric plant nuclei J95

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OBSERVATIONS

Examination of semithin longitudinal sections of Raphanus sativus root meristemshows that the size and shape of the interphase nuclei differ noticeably as a functionof distance from the apex. In order to facilitate our study and to be able to comparethe gross morphological features of nuclei at different stages of the mitotic cycle,observations were restricted to cells localized within the first millimetre of the root tip.

Fig. 4. Electron micrograph of portion of an early Gx nucleus slightly more advancedthan that illustrated in Fig. 3. Most of the nuclear cavity is occupied by a large, irregu-lar nucleolus consisting of compact heterogeneous central zones surrounded by looser,granular material (gm). Part of this latter material undoubtedly corresponds to rem-nants of the prenucleolar substance which exhibits a similar texture under electronmicroscopy and pervades the nuclear cavity throughout telophase (Fig. 2). A densechromosome segment (ch) is seen projecting through the nucleolar mass. The lightnucleoplasmic zones consist of loose, fibrillar material part of which presumably corre-sponds to diffuse chromatin. x 44 300.

Telophase and G1 nuclei

As is generally the case for chromocentric plant species (Delay, 1948; Singh & Roy,1974), the anaphase chromosomes of Raphanus sativus are only a couple of microns inlength and, under visible or ultraviolet optics, show few recognizable structuraldetails apart from the fact that their centromeric regions appear somewhat denser

Ultrastructure of chromocentric plant nuclei 197

than their more distal portions (Fig. 1). It is difficult, however, from the semithin sec-tions used for the present study to verify whether this apparent increase in densityof the centromeric regions is real or results simply from the sharp bending of thechromosomes at these places.

Subsequent to reaching the cell poles, the chromosomes get closer to one anotherwithin the small, irregular, forming nucleus, but maintain their rod-like, compactorganization. Following counterstaining with methylene blue a less-dense substance,the socalled prenucleolar material, is seen pervading the interchromosomalspaces, thus giving the early telophase nucleus a rather heterogeneous appearance(Fig. 2).

On account of the small size of the telophase nuclei and of the masking effects of theintervening prenucleolar material, their transition into interphase is not easilyanalysed. Judging mostly from the examination of series of consecutive semithin pre-parations it is evident, however, that from mid to late telophase large portions of thecondensed chromosomes transform into a diffuse state, with the result that only afew heteropyknotic chromatin masses remain distinctly visible in Feulgen prepara-tions. Concurrent with this structural evolution of the telophase chromosomes,the prenucleolar substance gradually disappears, thus giving rise to more trans-parent nucleoplasmic zones (Fig. 3). At the time when only small patches ofinterchromatin material remain, 2 small spherical bodies, the emerging nucleoli, maybe recognized. It is most likely from our electron-microscopic observations that the2 forming nucleoli are initially masked by the surrounding prenucleolar substance(Fig. 4) and have thus been growing for a while, or have sometimes even alreadyfused into a single mass, before they can be recognized as distinct structural entities.Nuclei which show such small nucleoli (Figs. 3, 4) are assumed to correspond to theearly Gj-period (Lafontaine & Lord, 1974ft).

Throughout the G1-period, the nucleolus increases markedly in size and eventuallyoccupies a large proportion of the nuclear cavity. In appropriate preparations, thisorganelle may be seen to be intimately associated with one or two dense chromatinmasses which are located close to the nuclear envelope. All other dense chromatinstructures are likewise found at the periphery of the nucleus. On account of theirintense staining with the Feulgen procedure, their compact ultrastructural organiza-tion and, as will be seen shortly, their particular labelling pattern, these chromatinmasses will henceforth be assumed to be chromocentric or heterochromatic in nature.The remaining transparent portions of the nuclear cavity show no structural organiza-tion under ultraviolet optics or following staining by means of the Feulgen-methyleneblue procedure. At the ultrastructural level, these nucleoplasmic areas are found toconsist of a fine, loose, fibrillar material within which it is impossible to recognizeany precise organization.

Nuclei of the S-period

Apart from the G1 nuclei just described, radish root meristems also exhibit a wholespectrum of other nuclei of similar geometry and chromatin organization but of largersizes. Labelling with tritiated thymidine permits one to recognize that certain of these


nuclei belong to the 5-period. Fig. 5, for instance, illustrates 8 adjoining nucleidiffering very little in their general morphological characteristics except for the factsthat 4 of them are slightly smaller and their chromocentres somewhat less con-spicuous than is the case for the remaining nuclei. Judging from the still irregularwalls separating the former cells, their nuclei are presumably at an earlier period ofinterphase than the others. Examination of the consecutive section (Fig. 6) which hasbeen processed for radioautography indeed shows that these 4 contiguous nuclei arethe only ones noticeably labelled, thus indicating that they most likely belong to thefirst portion of the early 5-period, whereas the larger nuclei have reached a later stage.

On account of the increased resolution furnished by electron microscopy, thepattern of labelling of S nuclei can be analysed in much greater detail and allowsclassification of these latter into 3 major groups. In a first group of nuclei, the radio-autographic grains are scattered rather uniformly over the loose fibrillar materialfilling the nuclear cavity, very few of them, if any, being localized over the peripheralheterochromatic masses or chromocentres which characterize this species (Figs. 7, 8).In this first category of nuclei, the nucleoli also exhibit a certain degree of labellingdirectly over or in the immediate neighbourhood of their lacunar areas. Althoughone or two chromocentres are often seen projecting within the nucleolar mass and,as in other plant nucleoli (Lafontaine & Lord, 19746), are assumed to be con-tinuous with diffuse intranucleolar chromatin, they do not show any radioautographicgrains (Fig. 8).

The second main group of nuclei which become labelled following 15-60 minincorporation periods also exhibit silver grains over the lacunar nucleolar regions,the remaining grains being mostly concentrated within the outermost portion of thenuclear cavity. Depending on the nucleus, this labelling may either be rather diffusethroughout the peripheral nuclear zone or take the form of clusters of grains overthe chromocentres (Fig. 9).

In the last group of nuclei, no radioautographic grains are observed over the nu-cleolar lacunae, only a few are over the diffuse nucleoplasmic fibrillar zones, mostof the labelling being now restricted to the chromocentres (Figs. 10-12). In many ofthese nuclei, the chromocentres exhibit a very loose organization and, following15- or 30-min labelling periods, the radioautographic grains are scattered uniformlyover their mass (Fig. 10). When roots are exposed to tritiated thymidine for 1 h some

Fig. 5. Micrograph of 8 adjacent Raphanus sativus nuclei which differ very little in theirgeneral morphological characteristics. The 4 nuclei on the left are, however, slightlysmaller than the others and their chromocentres are somewhat less conspicuous.Judging from the still irregular walls separating the former cells, their nuclei arecertainly at an earlier period of interphase than the others. Ultraviolet micrograph(260 ran) of a o-j-fim section of material embedded in glycol methacrylate. x 2800.Fig. 6. Micrograph of a section immediately adjacent to the one illustrated in Fig. 5and processed for radioautography. Incorporation of tritiated thymidine was carriedout for a 15-min period. Only the 4 nuclei on the left are noticeably labelled, thusindicating that they belong to the early or mid S-period whereas the 4 larger nuclei onthe right have presumably reached a later stage of interphase. x 2800.

Ultrastructure of chromocentrtc plant nuclei

* • # •


of the peripherally labelled nuclei exhibit compact chromocentres over which all thesilver grains are localized (Fig. n ) . A similar restriction of labelling to compactchromocentres is likewise observed in certain nuclei of roots exposed to tritiatedthymidine for only 15 min but subsequently transferred to water for 1-4 h (Fig. 12).

Nuclei also exhibiting irregular contours and gross morphological features verysimilar to those in the 5-period were identified as G2 nuclei on the basis of the follow-ing criteria: (a) these nuclei were not labelled; (b) they tended to be slightly largerthan most of the S nuclei; (c) their chromocentres were generally more conspicuous;and (d) such nuclei were also present in pairs separated by distinctly less wavy cellwalls than daughter cells with labelled nuclei (Figs. 5, 6). By the time nuclei reach thelate G2 or early prophase periods (Figs. 13, 14), they have greatly increased in size,their contours have become much more roundish than at earlier stages, and thechromocentres definitely begin to show a more elongate aspect as shown by earlierstudies of similar plant species (Doutreligne, 1933, 1939).

DISCUSSION

Owing to their small size and the fact that they are already partially masked by thesurrounding, socalled, prenucleolar substance, the structural evolution of Raphanussativus telophase chromosomes is most difficult to analyse. Earlier workers studyingsimilar plant nuclei have noted that the distal portions of chromosomes stain less andless intensely throughout telophase and that, eventually, only the centromeric regionsremain visible. Growth of the telophase nucleus is accompanied in these plants bymovement of the chromocentres to the periphery where they remain as distinctchromatin aggregates throughout interphase (Manton, 1935). The above variousobservations have led to the notions that, in such nuclei, the non-centromericchromosome segments transform into diffuse chromatin, whereas the centromericregions maintain their compact organization and correspond to the chromocentresobserved at interphase (Heitz, 1929; Doutreligne, 1933; Delay, 1948).

The lack of observable differentiation in the internal organization of radish inter-phase nuclei makes it difficult to determine whether they have reached the Glt Sor G2 periods. Our identification of the Gx and G2 nuclei is, therefore, mostly

Fig. 7. High-resolution radioautograph of an early S nucleus (Raphanus sativus) froma specimen labelled for a i-h period. The radioautographic grains are distributedrather uniformly over the fine, fibrillar material pervading the nuclear cavity. Sucha labelling pattern clearly demonstrates the presence of diffuse chromatin throughoutthe nucleoplasm. A restricted number of radioautographic grains is also observedover the nucleolus. x 28400.

Fig. 8. Electron micrograph of an early to mid-S nucleus after i-h labelling intritiated thymidine. As in the previous nucleus, the labelling is still uniformly dis-tributed over the diffuse chromatin filling the nuclear cavity. Three chromocentres(arrows) are visible in this plane of sectioning, none of which show any radioauto-graphic grains. Nucleolar labelling is slightly more appreciable than in Fig. 7, most ofthe silver grains being over the fibrillar zones, x 28000.

Ultrastructure of chromocentric plant nuclei

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based on rather gross criteria such as their size, the size of the nucleoli and the degreeof regularity of the new cell wall separating daughter cells. For the same reason, label-led nuclei are assumed to belong to the early, mid- or late 5-period depending on thedistribution of the radioautographic grains. We believe this labelling pattern variesin a sufficiently distinct fashion among S nuclei to furnish a reliable basis for sucha classification. Considering that euchromatin is well known to replicate first duringinterphase (reviewed in Moses & Coleman, 1964; Lima-de-Faria, 1969; Prescott,1970), those nuclei showing labelling over diffuse chromatin are taken to correspondto the early 5-period. The rather uniform distribution of radioautographic grains inthese nuclei reveals that euchromatin pervades extended regions of the nuclear cavityand must, therefore, represent a large portion of the chromosome mass. Taking intoaccount the appearance of radioautographic grains over the peripheral chromocentresand their decrease in number over diffuse chromatin, the second group of labellednuclei are undoubtedly more advanced than those just discussed and are assumed tobelong to the mid S-period. Similarly, in view of the restriction of labelling to thechromocentres in nuclei of the third group, it appears reasonable to hypothesize thatthese nuclei belong to the late 5-period, an assumption which also implies that thechromocentres are heterochromatic in nature (Heitz, 1929).

The finding that the degree of compactness of labelled chromocentres variesamong nuclei indicates that these structures undergo important modifications in con-formation as they replicate. Since radioautographic grains first appear over still com-pact chromocentres, initiation of DNA synthesis is most likely not accompanied byappreciable unravelling of these heterochromatic structures. The presence of labellingwithin the outer portion of certain of these chromocentres would rather, indeed,suggest that synthesis initially takes place at the periphery of these structures wherechromatin has possibly already relaxed. By late 5-period, however, there can be littledoubt that the chromocentres have transformed into much looser structures and thatDNA synthesis has attained a noticeably higher level of activity. Subsequently, asshown by our i-h or pulse-chase labelling experiments, chromocentres return to theirinitial compact organization, a conformation which they maintain during the G2-period. At prophase these heterochromatic structures are believed to act as foci ofchromosome condensation (Doutreligne, 1933, 1939; Delay, 1948).

Considered collectively, our observations reveal that the morphological evolutionand labelling pattern of plant chromocentric interphase nuclei present both

Fig. 9. High-resolution radioautograph of a mid to late S nucleus. In this nucleus,labelled for 15 min, a restricted number of radioautographic grains persist over thediffuse chromatin as well as over the nucleolus. All of the chromocentres visible inthis plane of sectioning show radioautographic grains, most of the latter beinglocalized at their periphery, x 22 500.Fig. 10. Portion of a mid S nucleus from a root which was labelled for 15 min and thentransferred to water for 30 min before fixation. In this radioautograph most of thesilver grains are restricted to 2 chromocentres both of which have taken a veryrelaxed conformation and, as a result, do not stand out very clearly against thesurrounding diffuse chromatin. x 30000.

Ultrastructure of chromocentric plant nuclei 203


similarities and differences with respect to the various animal and plant materials sofar studied. The unravelling of euchromatic chromosome segments during telophaseand concurrent appearance of diffuse chromatin throughout the nuclear cavity closelycorresponds, for instance, to the situation which generally prevails in animal cells(Erlandson & de Harven, 1971) but differs greatly from that observed in reticulateplant nuclei (Kuroiwa & Tanaka, 1970; Kuroiwa, 1974; Lafontaine & Lord, 1974a).The telophase movement of all heterochromatic chromosome loci to the peripheryof the nuclear cavity and their persistent attachment to the envelope during interphaseseems, however, to constitute a key characteristic of chromocentric nuclei (Manton,1935). Examination of serial sections of radish interphase nuclei shows that thenumber of heterochromatic structures is close to that of the chromosomes, a situationwhich has often been recorded in other plant species with chromocentric nuclei. Incertain of these plants all or most chromocentres are believed to originate from thecentromeres (Heitz, 1929; Doutreligne, 1933, 1939; Delay, 1948). Since our prepara-tions sometimes exhibit 2 chromocentres projecting within the nucleolar mass, itmust be inferred that these structures are not centromeric in nature but most likelycorrespond to heterochromatic chromosome segments adjacent to the nucleolarorganizer. Verification of the exact origin of the various chromocentres present inradish interphase nuclei would necessitate the study of metaphase chromosomes insquash preparations of material pulse-labelled with tritiated thymidine and processedfor radioautography (reviewed in Lima-de-Faria, 1969). Due to the small size of thechromosomes we have, unfortunately, not succeeded in obtaining sufficiently goodcytological preparations to undertake such studies. To our knowledge, the observedextensive relaxation of the late S chromocentre masses is a phenomenon which has,so far, been recorded only in other plant cell nuclei (Lafontaine & Lord, 1974a), eventhough labelling of late-5 animal cell nuclei is well known to be more appreciablewithin their peripheral portion where heterochromatin also tends to be localized(Blondel, 1968; Erlandson & de Harven, 1971; Huberman, Tsai & Deich, 1973).

A last observation which deserves discussion is the labelling of interphase nucleoli.In plant cells, this organelle has been shown to consist partly of an elongate filamen-tous structure, the nucleolonema (LaCour, 1966; Lafontaine & Lord, 1973, 19746),which is believed to correspond to the nucleolar organizer and to contain the ribo-somal genes. Although the nucleolonema of radish nucleoli is not particularly evident

Fig. 11. Electron micrograph of portion of a late S nucleus depicting the specificlabelling of the chromocentres at this period of interphase. Following incorporationof tritiated thymidine for 1 h, the radioautographic grains are distributed uniformlyover the mass of the chromocentres. The nucleolus no longer shows evidence ofincorporation of this DNA precursor, x 28000.Fig. 12. Portion of a nucleus from a root exposed to tritiated thymidine for 15 minand then transferred to water for 4 h. Since the nucleolus and diffuse chromatin areashow no significant labelling, incorporation of thymidine is assumed to have takenplace within the chromocentres during the late 5-period. At the time of fixation, thisnucleus may, therefore, have reached the Ga period. Each chromocentre is heavilylabelled and exhibits a compact organization, x 31000.

Uhrastructure of chromocentric plant nuclei

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12

206 A. Lord and jf. G. Lafontaine

13 14Fig. 13. Ultraviolet micrograph of Raphanus sativus nuclei which are considered tohave reached the G.-penod on account of their increased size, their more roundishcontours and the absence of labelling in a consecutive section processed for radio-autography. The chromocentres are quite conspicuous and are located at the nuclearperiphery, x 3400.Fig. 14. Ultraviolet micrograph of a nucleus showing structural changes characteristicof prophase in this species. The nucleolus has increased in size and its contours havebecome more angular. The chromocentres also begin to show a more elongate aspectas the chromosomes start condensing, x 3400.

as such under electron microscopy, due to insufficient density difference with thesurrounding nucleolar material, it can be visualized within the fibrillar zones of thenucleolus by means of phase-contrast optics. The presence of radioautographic grainsover the lacunar portions of these fibrillar zones in early and mid-5 nuclei may betaken, therefore, to reflect the replication of ribosomal DNA during the first half ofthe 5-period, a conclusion also recently reached in biochemical studies of animal cellsin culture (Amaldi, Giacomoni & Zito-Bignami, 1969; Stambrook, 1974).

The authors thank Mrs Diane Michaud, Claudette Milani and Mr Siegfried Gugg for theircapable technical assistance.

This work was aided by grants from the Quebec Ministry of Education and from theNational Research Council of Canada. The financial assistance of the Donner Canadian Foun-dation for purchasing the Zeiss UMSP-i instrument is also gratefully acknowledged.

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(Received 22 October 1975)

an ultrastructural and radioautographic study …examination of semithin longitudinal section...

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