An ultrastructural study of plant cell (Allium porrum) centromeres

JOURNAL OF ULTRASTRUCTURE RESEARCH 70, 298-307 (1980)

An Ultrastructural Study of Plant Cell (Allium porrum) Centromeres J. G. LAFONTAINE AND B. T. LUCK Ddpartement de Biologie, Facultd des Sciences et de Gdnie, Universitd Laval, Qudbec GIK 7P4, Canada Received July 16, 1979, and in revised form, October 29, 1979 A number of reports have appeared in recent years on the presence of fibrillar nuclear formations in plant cells. Owing to their intimate association with the chromosomes as well as to their ultracytochemical characteristics, these spherical structures have been assumed to represent centromeres. In the present study, serial sections of complete nuclei were examined in order to obtain more representative data on the number, size, and distribution of the centromeres during interphase. Less detailed but equally interesting information was also obtained on prophase nuclei. Our observations reveal that centromeres tend to be clustered in the polar portion of telophase nuclei but that they are displaced to some extent during interphase. Even during prophase, the dispersion of the centromeres is far from random. The fact that certain centromeres are two, four, and sometimes eight times more voluminous than others is taken to indicate that fusion of these structures takes place during interphase; this accounts for the observation that the number of centromeres is generally noticeably less than the total number of chromosomes. These large centromeric aggregates fragment during prophase and pairs of normal size centromeres are observed in association with the daughter late prophase chromatids.

Various electron microscopic studies of plant interphase nuclei have appeared in recent years describing formations, usually roundish in outlines, and consisting of fine fibrillar material. Although these fibrillar structures have been referred to as karyosomes (Hyde, 1966, 1967) and micropuffs (Lafontaine and Lord, 1969; Lafontaine, 1974; Risuefio et al., 1978), conclusive evidence obtained from the examination of serial sections was recently presented (Church and Moens, 1976; Moens and Church, 1977) to the effect that they correspond, in fact, to centromeres. Concurring additional data were also recently provided demonstrating that these fibrillar nuclear formations contained DNA, RNA, and proteins and therefore exhibited cytochemical properties similar to those expected of chromatin (Lafontaine et al., 1979). Other observations, such as their frequent intimate association with one or more of the dense chromatin strands which characterize A l l i u m p o r r u m interphase nuclei (Lafontaine and Lord, 1974a), were likewise interpreted in favor of the view that these nuclear formations represent specific chromosome segments.

Although several ultrastructural studies were undertaken on centromeres in plant cells undergoing meiosis, corresponding observations have not yet been carried out during the different stages of the mitotic cycle (reviewed in Bajer and Mol~-Bajer, 1972; Church and Moens, 1976). The present work was therefore undertaken, partly with the help of serial sections, in order to investigate the distribution, the number, as well as the size of these structures at interphase and prophase, stages of the mitotic cycle during which they are easily observed. For that purpose we selected a species (Allium porrum) the nuclear organization of which is well known (Lafontaine and Lord 1974a,b). The data obtained reveal that these puff-like fibrillar structures correspond to distended segments of chromosomes and, judging from their polar distribution in telophase and early interphase nuclei, they clearly are centromeric in nature. It is further observed that, although centromeres move to a certain extent during subsequent growth of the interphase nucleus, they do not appear to become completely scattered throughout the nuclear cavity. Small groups of five or six centro-

298 0022-5320/80/030298-10502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

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meres are, indeed, frequently observed not only during the various periods of interphase but also up to midprophase. It is also found that centromeres vary greatly in size during both interphase and prophase, presumably as a result of fusion. MATERIALS AND METHODS Root tips (Allium porrum) were fixed in 4% glutaraldehyde adjusted to pH 7.2 with sodium cacodylate and, after washing in buffer, were postfixed in 1% osmium tetroxide. Sections prepared from Eponembedded specimens were stained with both uranyl acetate and lead. citrate. Other root tips fixed in icecold 4% formaldehyde were stained, before embedding, with 3,3'-diaminobenzidine (DAB) in order ~to reveal nucleic acid-containing structures (Anteunis et al., 1973} and thus avoid further processing of the sections prepared from these specimens. For the present study, 212 consecutive sections from a DAB-stained root were mounted on Formvar-covered single-hole grids and were first examined at low magnification for recording selected areas. Higher magnification micrographs were finally obtained of the nuclei which showed relevant morphological information. These observations were carried out with a Philips EM 300 electron microscope. Volumes of the centromeres were estimated by measuring their surfaces in consecutive sections and then multiplying by 0.1 ~tm, the thickness of the preparations. OBSERVATIONS

Distribution of the Centromeres Earlier investigations showed that pufflike structures, now believed to be centromeres, were first recognized in early to midtelophase nuclei when the chromosomes, still aligned along the cell poles, began to slowly unravel (Lafontaine and Lord, 1969). At that stage the centromeres appeared as loose fibrillar structures 0.25-0.4 ttm in diameter. In the course of the present work, telophase centromeres were seen to be deeply embedded within the chromosomes and also to tend to cluster throughout the proximal portion of the forming nucleus. A similar distribution of the centromeres was observed in late telophase or early G1 nuclei (Fig. 1). Slightly later on, the chromosomes have unraveled considerably and they have transformed into narrow, irregular strands forming a complex chromatin reticulum

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(refer to Lafontaine and Lord, 1974a). As in telophase, groups of four to six centromeres were recognized, in a given section, within the proximal portion of the nucleus but they were then part of the chromatin reticulum and were, therefore, generally intimately associated with two or more chromatin strands (Fig. 2). During the G1 period, the four nucleoli increased considerably in size and some of the centromeres were then observed within the median portion of the nucleus and even sometimes in regions just distal to the nucleoli (Fig. 3). Although examination of individual sections suggested that the centromeres became still more scattered during subsequent growth of the nucleus, data obtained from serial preparations showed that, in S and G2 nuclei, centromeres were not distributed at random but still tended to be grouped within a portion of the nuclear cavity. At early prophase, loose, spherical structures corresponding to centromeres were recognized in close association with irregular profiles of the chromosomes or sometimes more or less embedded within some of them (Fig. 5). By midprophase, groups of centromeres were still detected, certain of them being very close to one another to form pairs which appeared to be associated with daughter chromatids. Other fibrillar structures were oblong in shape and seemed to consist of at least two fused centromeres (Fig. 6). When midprophase chromosomes were sectioned transversely, certain centromeres were found to be distinctly located between the two paired chromatids (Fig. 7). The characteristic grouping of several centromeres in one portion of the interphase and prophase nucleus, referred to above, was observed to persist till at least the beginning of late prophase (Fig. 8). At the time the nucleolus breaks down, however, the late prophase chromosomes have become quite compact in organization and centromeres were not often detected. No special structures were observed during metaphase and anaphase that could be

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Fro. 1. Electron micrograph of a late telophase nucleus (Alliumporrum). The chromosomes which are just beginning to unravel exhibit a vacuolated structure and are seen to consist of contorted filamentous subunits. In this plane of sectioning, six loose fibrillar structures may be detected in close association with the chromosomes (arrows). Judging from their size, shape, and, for some of them, their proximity to the still most irregular nuclear envelope, these puff-like formations correspond to centromeres. Material fixed with glutaraldehyde-osmium tetroxide and stained with uranyl acetate-lead citrate. × 21 000. FIG. 2. By the mid-G1 period, the chromosomes have undergone extensive unraveling and have thus given rise to a complex chromatin reticulum characteristic of plant interphase nuclei such as those of Allium porrum. At that stage, the growing nucleoli are predominantly made of fibrillar material. The centromeres are slightly denser than in earlier stages and are consequently more easily recognized. It should be noted, most particularly, that each centromere, even those closely associated to the nuclear envelope, is linked to several small chromatin masses. In the case of the latter centromeres it is clear, moreover, that they are not directly attached to the nuclear envelope but rather by means of the associated chromatin. Material processed as in Fig. 1. × 21 000. FIG. 3. Portion of a G~ nucleus in material fixed with 4% formaldehyde and stained with DAB in the block. The nucleoli do not show the usual zonation into granular and fibrillar areas in such specimens (authors' unpublished observations). The chromatin strands, however, stain as densely as in conventional preparations. Puff-like structures, presumably centromeres (arrows), may be recognized in the upper portion of the nucleus. Several of these are in the neighborhood of the nucleolar surface and have been displaced from their original polar localization by the growing nucleolus. × 23 000. easily identified as centromeres although V-shaped regions of mitotic chromosomes in serial sections were carefully examined.

A s s o c i a t i o n o f the Puff-like S t r u c t u r e s w i t h the C h r o m a t i n S t r a n d s Even though the loose fibrillar structures

described in this paper are now assumed by most workers to correspond to centromeres, there does not yet exist a concensus on this et al., matter (discussed in Lafontaine 1979). A c c o r d i n g l y , s p e c i a l a t t e n t i o n w a s given during the examination of serial sections to those puff-like bodies which either

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appeared to lie completely free in the nucleoplasm or seemed to fortuitously rest against a chromatin strand. It was thus found that all these fibrillar structures are, in fact, intimately associated with a minimum of two chromatin strands at interphase (Fig. 4). Seemingly free spherules within the nucleoplasm were also easily verified to represent tangential sections of chromatin-associated structures. Variation in the Size and Number of Centromeres Besides permitting demonstration of the chromosomal nature of the puff-like formations, the examination of serial sections also furnished useful data on their number and size in different nuclei. Observations carried out on five complete interphase nuclei revealed that three of them contained 15 centromeres, a number which is about half that of the chromosomes (2n = 32) in this tetraploid species (Levan, 1931). Only 11 and 12 centromeres, respectively, were present within the other two nuclei. It was also noted that the diameter of these globular structures sometimes varied considerably within a nucleus. Most important, perhaps, was the finding that, within a given nucleus, the volumes of certain centromeres were approximately two, four, and even eight times larger than those of the smallest ones (0.05 ~m3). Unfortunately, similar data could not be obtained during prophase for lack of a series of sufficient consecutive sections to represent entire nuclei. It was nevertheless evident from certain micrographs (Fig. 6) that some centromeres were noticeably larger than others.

DISCUSSION

Centromeric Nature of the Puff-like Nuclear Formations The fibrillar globular formations ohserved in Allium porrum interphase nuclei are basically similar in ultrastructure to the centromeres identified in both meiotic (Dietrich, 1968; Wilson, 1968; Braselton and Bowen, 1971; Gillies, 1973; Moens and Church, 1977) and mitotic plant cells (Bajer and Mol~-Bajer, 1972; Church and Moens, 1976). As expected of specific chromosome segments, these structures have been shown to display ultracytochemical characteristics quite similar to those of chromatin (Lafontaine et al., 1979). In the course of the present investigation it was possible, by means of serial sections, to establish that the smallest of these puff-like bodies are intimately associated with two neighboring chromatin strands (Figs. 4a-f), whereas the bigger ones are attached to several strands. Such findings are at odds with the notion recently held by some investigators that these fibrillar formations are of nucleolar origin and lie free in the nucleoplasm (Risuefio et al., 1978). We have not found any evidence supporting the view presented by these latter authors that certain of these puff-like nuclear formations moved across the nuclear envelope into the cytoplasm. Position and Movement of the Centromeres The fact that, in certain organisms, interphase chromosomes maintain a nonrandom arrangement throughout interphase has

FIG. 4. Micrographs of a group of three centromeres recorded in six consecutive sections. Each centromere appears heterogeneous due to the presence of slightly clumped fibrillar portions which stain just as densely as the chromatin strands. It is also noted t h a t the size of these centromeres varies noticeably. The smallest one, best seen in the left portion of a, is approximately 0.05 t~m3 in volume; the largest centromere which appears in all six micrographs is roughly eight times more voluminous. Examination of these micrographs reveals that all centromeres are intimately associated with chromatin formations. It may easily be verified, moreover, that a larger n u m b e r of chromatin strands are linked to the bigger centromere. Material stained according to the DAB technique. × 26 000.

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FIG. 5. Portion of an early prophase nucleus from specimen stained with DAB. As in interphase, the chromosomes react intensely with this stain. At that stage, however, owing partly to the irregular contours of the coiling chromosomes, centromeres are somewhat more difficult to detect. In this micrograph three looser regions of the chromosomal strands may be recognized as corresponding to centromeres {arrows). x 26 000. FIG. 6. Portion of a midprophase nucleus depicting the coarse ultrastructural organization of certain rather large centromeres sometimes observed at this stage. Specimen stained with DAB. x 30 000. Fro. 7. Part of a midprophase nucleus illustrating a cross-section of chromosome which clearly consists of two paired chromatids. The centromere (arrow), interestingly enough, appears to be located between the daughter chromatids (ch). Unfortunately, it was not possible to verify whether the centromere itself is double. Specimen stained with DAB. x 31 000. Fro. 8. View of portion of a late prophase nucleus. On account of the difficulty of visualizing centromeres at that stage, the specimen was treated with ribonuclease for 2 hr before staining in the block with DAB. In such preparations the centromeres take on a more transparent appearance, presumably as a result of extraction of a RNP matrix (Lafontaine et al., 1979) and are, therefore, more easily recognized. At least 13 centromeres (arrows) are visible in this region of the nucleus, thus suggesting that their displacement is not random during interphase and prophase. It could just as convincingly be argued, perhaps, that this grouping of the centromere reflects movement of these chromosome regions toward one portion of the nucleus in preparation to their eventual attachment to the spindle microtubules. Two of the centromeres {lower right corner) are closely appressed and seem to belong to daughter chromatids, x 16 500. b e e n k n o w n f r o m a n u m b e r o f classic obs e r v a t i o n s ( d i s c u s s e d in W i l s o n , 1925; S w a n s o n , 1957). I n t h e case of p l a n t s (Allium cepa), p a r t i c u l a r l y d e t a i l e d l i g h t microscopical observations were carried out b y H e i t z (1932) a n d F u s s e l (1975, 1977). Using radioautographic and C-banding

techniques, this latter author has furnished e v i d e n c e of t h e p e r s i s t e n c e d u r i n g i n t e r phase and prophase of the centromeric clustering which characterizes telophase. E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s o f Al-

lium fistulosum m i c r o s p o r e s h a s r e v e a l e d , h o w e v e r , t h a t c e n t r o m e r e s do n o t m a i n t a i n

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their initial position within the interphase nucleus but become relocated with respect to the nuclear envelope (Moens and Church, 1977). In the case of Allium porrum meristematic cells studied in the course of the present work, the data obtained clearly show that the telophase arrangement of the centromeres is not rigidly maintained during interphase and that most of these structures, indeed, move away from the nuclear envelope. Nevertheless, the fact that groups of five or six centromeres were often observed throughout interphase as well as during prophase indicates that the displacement of these specific chromosomes loci is not random.

Variation in the Size of the Centromeres Recent ultrastructural investigations (Church and Moens, 1976; Moens and Church, 1977) have shown that the size of centromeres varies in both mitotic and meiotic plant cells (Allium fistulosum). No volume determination was carried out by these authors in meristematic interphase nuclei but the volume of individual centromeres in premeiotic interphase nuclei was found to range from 0.045 to 0.353 ttm3. It was concluded from such data, as well as from the observed number of centromeres per nucleus, that association occurred between two and up to about seven of these structures during interphase. This interpretation was supported by the additional finding that the larger fused centromeres were embedded within proportionally more conspicuous aggregates of centric heterochromatin, or chromocenters. The situation which prevails in Alium porrum is generally quite similar to that just discussed. The volume (0.05 ~m 3) of the smallest centromeres observed at interphase in this species is, indeed, only about twice that of those seen at mitotic prophase and roughly the same as that of those recorded at meiotic interphase in Allium fistulosum. Such similarity in size may well indicate that the basic dimension of centromeres is more or less constant, at least in

related plant species. As hypothesized by these authors our data, likewise, suggest that the larger centromeres represent aggregates of two or more basic units which, in Allium porrum, are approximately 0.05 /~m3 in volume. Since certain centromeres appear to consist of eight smaller ones, it must be further assumed that their association during interphase is not necessarily homologous. Our observations also show that centromeres tend to be more constant in size by midprophase thus suggesting that the larger centromeric associations seen earlier disperse during that stage into individual structures. This phenomenon undoubtedly accounts for the presence of pairs of centromeres closely associated to daughter midprophase chromatids. A plausible explanation of the foregoing observations is that dissolution of the composite centromeres constitutes a prerequisite to the eventual movement of the late prophase chromosomes. The authors wish to thank Dr. P. Moens for his advice in the preparation of serial sections. We are also particularly grateful to Mr. S. Gugg who was responsible for preparing these sections as well as for the measurements contained in this paper. Financial assistance was provided by the Natural Sciences and Engineering Research Council Canada and the Quebec Ministry of Education. REFERENCES

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(1979) J. Cell Sci. 39, 13-27. LEVAN, A. (1931) Hereditas 15, 347-356. MOENS, P., AND CHURCH, K. (1977) Chromosoma 61, 41-48. RISUEI~O, M. C., MORENO DIAZ DE LA ESPINA, S., FERNJ~NDEZ-G6MEZ,M, H., AND GIMI~NEZ-MARTIN, G. (1978). Cytobiologie 16, 209-223. SWANSON, C. P. (1957) Cytology and Cytogenetics, Prentice-Hall, Englewood Cliffs, N.J. WILSON, E. B. (1925) The Cell in Development and Heredity, 3rd ed., Macmillan, New York. WILSON,H. J. (1968} Planta 78, 379-385.