THE RELATIONSHIP BETWEEN NUCLEAR DNA CONTENT AND CENTROMERE VOLUME IN HIGHER PLANTS

Transcription

1 J. Cell Sci. 47, (1981) 91 Printed in Great Britain Company of Biologists Limited KJSI THE RELATIONSHIP BETWEEN NUCLEAR DNA CONTENT AND CENTROMERE VOLUME IN HIGHER PLANTS M. D. BENNETTf, J. B. SMITH, J. WARD AND G. JENKINS* Plant Breeding Institute, Trumpington, Cambridge CBz LQ, England SUMMARY The total volume of centromeres per nucleus varies widely within Triticum aestivum cv. Chinese Spring (7-fold) and between 11 higher plant species (4-fold). Such variation is closely correlated with nuclear DNA content, nuclear volume and, to a lesser extent, the total volume of nucleoli per nucleus. Centromere volume reflects minor intraspecific developmental fluctuations in nuclear size independent of variation in nuclear DNA content, but variation in nuclear DNA plays the major role in determining centromere volume. Thus, in general a given total volume of centromeric material is apparently characteristic of an approximately constant nuclear volume and mass of nuclear DNA, but largely independent of chromosome number. The range of volumes of single centromeres in 4 taxa corresponds with the ranges of their single chromosome lengths or chromosome DNA contents. The centromere is, therefore, not a unit structure of constant size and mass but a chromosome segment whose highly variable volume closely reflects the volume and mass of the chromosome to which it belongs. The correlation between centromere size and chromosome size and DNA content is potentially useful for identifying single centromeres in unsquashed interphase and dividing nuclei; thereby facilitating studies of the intranuclear disposition of chromosomes. The present results for centromeres provide probably the first example to indicate that variation in the total DNA content of small segments present on each chromosome sometimes varies directly in proportion to large interspecific variation in nuclear DNA C-value. The close correlation between centromere volume, and nuclear DNA content is probably nucleotypic in origin. The functional significance of the variation in centromere volume is unknown, as is the nature of the mechanism which determines that centromere volume closely reflects nuclear and chromosome size and mass. INTRODUCTION The 4C DNA amount varies widely between higher plants (Bennett & Smith, 1976), as does the total volume of chromosomes per \C nucleus at mitosis (Rothfels & Heimburger, 1968). Several studies have shown that \C DNA amount and total chromosome volume are almost directly proportional when species of the same genus are compared (Rees, Cameron, Hazarika & Jones, 1966; Pegington & Rees, 1970). Chromosomes are seen to comprise segments of various sizes, including chromomeres, primary and secondary constrictions and heterochromatic knobs or bands. In view of the close relationship between nuclear DNA content and total chromosome volume it is interesting to question whether interspecific variation in the volume of particular Agricultural Botany Department, University College of Wales, Aberystwyth SY3 3DD, Dyfed, Wales. f To whom reprint requests should be sent.

2 9 M. D. Bennett and others small chromosome segments sometimes reflects the directly proportional relationship with nuclear DNA content shown by the whole complement of which they form a part. Instances are known in which variation in nuclear DNA content involved disproportionately large increases in the size and DNA content of heterochromatic segments on one or several chromosomes within the complement. For example, in Secale a difference in 4C DNA content of about 0 % is largely accounted for by about a doubling in the size (Bennett, Gustafson & Smith, 1977) and in the DNA content (Bedbrook et al. 1980) of segments of telomeric heterochromatin in the complement. Moreover, variation in the amount of heterochromatin in the genus Secale was very unequal between homoeologous chromosomes (see fig. 1 in Bennett et al. 1977). As far as the present authors are aware, no example is known in which changes in the volume or DNA content of small segments, present on many or all of the chromosomes in a complement, are directly proportional to variation in either the total volume or the DNA content of the complement as a whole. Centromeres contain DNA, but normally at a significantly lower density than most other chromosome segments in higher plants (as judged, for example, by the relatively poor Feulgen staining of primary constrictions). Moreover, the centromere is a small chromosome segment easily recognized in electron micrographs of several higher plant species (Gillies, 1973; Church & Moens, 1976). It is possessed by each chromosome in the complement and shares a common function at karyokinesis. Thus, the centromere represents an ideal subject for an investigation of possible relationships between the size of a small defined chromosome segment and interspecific variation in nuclear size and DNA content. The pioneering work on centromeres in AUiumfistulosum (Church & Moens, 1976; Moens & Church, 1977) showed that their total volume per nucleus can vary considerably, both during the cell cycle and between different tissues. As information concerning intraspecific variation in centromere volume was available for only one plant species, similar studies for a second species seemed worthwhile, and Triticum aestivum cv. Chinese Spring was chosen. It was decided to estimate other nuclear-size characters also, in an attempt to understand variation in centromere volume in the context of the nuclear environment. MATERIALS AND METHODS The materials used in the present work were all available at the Plant Breeding Institute (apart from the Festuca hybrid), and are listed in Table 1. The Festuca hybrid was kindly supplied by Drs M. Borrill and W. G. Morgan of the Welsh Plant Breeding Station, Aberystwyth, where it was grown outside. Otherwise plants were cultivated in a growth room at 0 ± 1 C with continuous light for at least several days before tissue was sampled. Root-tip or anther material was usually sampled and prepared for electron microscopy as previously described (Bennett, Smith, Simpson & Wells, 1979). However, in a few instances (marked with an asterisk in Tables, 3, pp. 98, 100), instead of double staining with uranyl acetate and lead citrate, material was stained only with phosphotungstic acid in absolute ethanol for about 18 h, as described by Sheridan & Barnett (1969). Serial sections about 100 ran thick were cut using a diamond knife on a Reichert Ultracut IV microtome, and collected on Formvar-coated, x1 mm slot-grids, using the technique of Wells (1974). Selected nuclei

3 Nuclear DNA content and centromere volume 93 were photographed at x 4500 using a Philips 01 electron microscope at 80 kv, and printed at a final magnification of x Centromeric structures and nucleoli were traced from each photomicrograph on which they appeared on to sheets of polyester film of constant thickness using a fine-tipped pen with waterproof ink. However, nuclei were traced from every fifth section. Plastic sheets of tracings were photocopied for a permanent record, before the tracings of each selected structure were cut out by hand, using scissors, and weighed. An ii-8-cm square of polyester sheet was also weighed, corresponding to a structure 10 ^m 8 in volume. The weight of tracings of each selected structure was converted to fim* by comparison with the weight of this standard square. Complete series of photomicrographs were obtained for 53 nuclei, including 31 from 10 different tissues or stages of development in Triticum aestivum (85-0 sections) and meiocytes ( sections) at early premeiotic interphase or the first prophase in 10 other species. Table 1. Chromosome number (/1), ploidy level (x) and \C DNA amount (from Bennett & Smith, 1976) in 14 taxa used in the present work Taxon zn X 4C DNA amount, pg Dicots Crepis capillaris Vicia faba Monocots Briza maxima Cicer arietimtm Eragrostis tef Festuca scariosa x drymeja Hordeum bulbosum Hordeum vulgare Secale cereale Tradescantia paludosa Triticum monococcum Triticum dicoccum Triticum aestivum Zea mays 6 1 H '3 43' O '4 RESULTS Centromere identification and DNA amount per chromosome Centromeric structures were recognized in the present work by their gross appearance, ultrastructure and intranuclear distribution. During interphase and leptotene they appeared as light staining spheres of fibrils embedded in a meshwork of darker staining chromatin (Fig. 1). During zygotene and pachytene they were usually elongated in the long axis of the synaptonemal complex or lateral element running through them (Fig. ). At interphase all centromeric structures were found near together at one side of the nucleus in a relic telophase configuration, while at first prophase of meiosis they were distributed more widely within the nucleus. Thus, the structures taken to be centromeres were identical in every respect with those described previously in higher plants at these stages (Church & Moens, 1976; Moens & Church, 1977), and unequivocally shown to be centromeres in Zea mays (Gillies, 1973). Further evidence of correct identification comes in the present work from the observation that, at first metaphase in Triticum aestivum, each bivalent possessed two

4 M. D. Bennett and others *, Fig. i. Lighter-staining CeSs (arrowed) and darker staining chromatin in a Chinese Spring meiocyte nucleus at early premeiotic interphase (stage s, replicate a in Table 3). Bar, 1 /(m. Fig.. Lighter-staining CeS (arrowed) elongated in the plane of the lateral component of the synaptonemal complex running through it in a Chinese Spring meiocyte nucleus at zygotene (stage 8, replicate in Table 3). Bar, 1 /an.

5 Nuclear DNA content and centromere volume 95 such structures, both with attached spindle microtubules (Fig. 3), one at either end. The term 'centromeric structure' (CeS) as defined by Church & Moens (1976) is used in the present work to describe both single centromeres and associated groups of or more centromeres. Detailed results of the intranuclear distribution and association of centromeres will be presented elsewhere. Fig. 3. Lighter-staining centromeres (arrowed) at either end of a bivalent in a Chinese Spring bread wheat meiocyte at first metaphase (stage 9, replicate in Table 3). Bar, 1 fim. Fig. 4. Typical thin-section through a serially sectioned meiocyte nucleus (arrowed) of Eragrostis tef at premeiotic interphase showing very little condensed chromatin. Bar, 1 fim. Although anthers were fixed at one or more stages from early premeiotic mitosis until pachytene, most anthers of the species listed in Table 1 were fixed at early premeiotic interphase, because nuclei were smaller in diameter at this stage and hence

6 96 M. D. Bennett and others required fewer sections for their complete reconstruction. Of the 11 species for which material at early premeiotic interphase was available, centromeres of similar appearance were readily distinguished in all except Cicer arietinum, Eragrostis tef and Zea mays. The mean 4C DNA content per chromosome in the 8 species in which centromeres were distinguished ranged from 0-96 pg (in the Festuca hybrid) to 4-4 pg (in Vicia faba), while in the three species where centromeres could not be distinguished it ranged from 0-07 (in Eragrostis tef) to 0-47 pg (in Zea mays). Thus, recognition of CeSs at early premeiotic interphase using the present techniques is apparently related to the mean DNA content per chromosome; between about 0-5 and 0-9 pg of DNA per single chromosome in 4C nuclei being required before centromeres can be regularly and reliably identified. Presumably CeSs in species whose mean DNA amount per chromosome is below this threshold might still be identified at other developmental stages, since for example, centromeres of Zea mays (not distinguished at early premeiotic interphase in the present work) were easily distinguished, when paired, at pachytene (Gillies, 1973). The striking differences between the characteristic appearance of nuclei in plant species with low, intermediate or high 4C DNA amounts, seen in both the light and electron microscopes, have been noted previously (Barlow, 1977; Nagl, 1979). In species with the lowest 4C values (which display the areticulate form) the chromatin reticulum is more or less invisible in the light microscope, and so dispersed or decondensed as to render defining its exact extent difficult or impossible in the electron microscope (Fig. 4). In general, the proportion of the nucleus occupied by visibly condensed chromatin masses increases with increasing \C DNA amount (especially over the range 0 0 pg); species with high C values have more DNA per unit nuclear volume than species with low C values (Sunderland & McLeish, 1961). Thus, the present results for centromere recognition agree with previous general observations of plant chromatin. Interspecific variation in centromere volume Table gives the total volume of CeSs, the total volume of nucleoli, and the nuclear volume for 6 nuclei at either early premeiotic interphase or first prophase of meiosis, in 11 higher plant species. Overall there was about a 4-fold variation for estimates of the total volume of CeSs per nucleus ranging from 0-65 (in a Crepis capillaris nucleus at early premeiotic interphase) to 15-6 fim 3 (in a Tradescantia paludosa nucleus at zygotene). The total nuclear and chromosomal DNA content at first meiotic prophase (4C) is twice that at early premeiotic interphase (C), while, in Triticum aestivum and T. monococcum (see Results, p. 97), the total volume of CeSs at first meiotic prophase is approximately double that at early premeiotic interphase. It is therefore permissible, and more meaningful, to compare the total volume of CeSs per C DNA amount, by halving centromere volume in 4C meiotic nuclei. Having done this, estimates of the total volume of CeSs show about a 1-fold range, from 0-65 in Crepis capillaris to 7-8 fim 3 in Tradescantia paludosa. The overall variation in nuclear volume was about i4'7-fold, from a minimum of 160 fim 3 in Crepis capillaris to fim 3 in Tradescantia paludosa. The nuclear

7 Nuclear DNA content and centromere volume 97 volume per zc nuclear DNA amount (obtained, where appropriate, as described above), differed about io-8-fold, from 98-3 /tm 3 in a nucleus at zygotene in Festuca hybrid, to /im 3 in Triticum aestivum at early premeiotic interphase. Total nucleolar volume was somewhat less variable overall than either total centromere volume, or nuclear volume. Thus, there was only about a 7-6-fold range from a minimum of 1-7 in Hordeum vulgare at very early premeiotic interphase, to 96-4 /tm 3 in Triticum aestivum. Examination of the data in Table shows that the ranges of variation between cells of the same species for estimates of total volume of CeSs (1-44-fold), nuclear volume (i-58-fold) and nucleolar volume (i-7-fold) were small compared with the interspecific ranges for estimates of these 3 characters. Intraspecific variation in centromere volume and other characters Table 3 presents estimates for the total volume of CeSs per nucleus, the total nucleolar volume, and the nuclear volume in 31 completely sectioned nuclei of Chinese Spring sampled at 11 different developmental stages in root-tips or anthers (including 4 nuclei at early premeiotic interphase from Table ). The total volume of CeSs in single nuclei varied about 7-fold, ranging from -36 in a young microspore to /im 3 in a zygotene meiocyte. Nuclear volume also varied by about 7-fold, ranging from (in the same young microspore) to 460 /tm 3 (in the same zygotene meiocyte). By comparison, total nucleolar volume varied about 8-5-fold, ranging from 13-9 in an anther connective somatic cell to 118-7ft - 3 m the zygotene meiocyte. In Chinese Spring development is approximately synchronous between nuclei within an anther at stages from early premeiotic interphase to young microspores inclusive, and premeiotic DNA synthesis occurs during mid- to late premeiotic interphase (Bennett, Rao, Smith & Bayliss, 1973; Bennett et al. 1979). The total volume of CeSs per nucleus was similar within (but not between) samples of 4 zc nuclei at early premeiotic interphase (range /im 3 ), 3 ic nuclei from young microspores (range /mi 3 ) listed in Table 3, and in another sample of 5 ic nuclei from young microspores of Chinese Spring (not included in Table 3) at a slightly later stage of development which had total centromere volumes per nucleus of -79, 3-0, 3-07, 3-1 and 3-30 /tm 3. The present results agree, therefore, with those for Allium fistulosum (Church & Moens, 1976) in showing much less variation in total volume of CeSs between approximately synchronous replicate nuclei at a single developmental stage, than between samples of nuclei at different stages. Total centromere volume per nucleus and chromosome number (zn) Chromosome number {zn) differed 7-fold in the 11 species listed in Table, ranging from 6 (in Crepis capillaris) to 4 (in Triticum aestivum). Table shows first that both nuclear DNA content and total volume of CeSs per nucleus varied widely between species with the same chromosome number (zn = 14). Secondly, Viciafaba (6-65 pg) and Triticum dicoccum (4-55 pg) have similar zc DNA amounts but different chromosome numbers (1 and 8, respectively). Table shows that the

8 M. D. Bennett and others

9 Pachy. I I o. Tritinmt diccaum Epi. I 3 Mean I I. Tn'ticum aestivum and known DNA content (DNA) of nuclei at early premeiotic interphase (epi.), leptotene (lept.), zygotene (zygo.) or pachytene (pachy.) 5 Epi. I 3 4 Mean b The estimated nuclear volume (n.v.), total centromere volume (~c.v.), and total nucleolar volume (znlr.~.), and the chromosome number (zn) n 0 in 11 species, together with derived estimates of the proportion of the total nuclear volume occupied by centromeres exprased as a percentage 3 (zc.v./n.v. %), the mean centromere volume per chromosome (%.v./zn), the mean DNA content per chromosome (DNA/zn), and the volume of centromeric material per pg of nuclear DNA (Cc.v./DNA). The number of sections per nucleus is also given. (N.B. An asterisk in column 3 3 indicates a nucleus stained with phosphotungstic acid.) a. 3 P 0

10 M. D. Bennett and others * PP e Po? F!' $$ o o mcn t l n - N H w I I I I I I I I I I I 1 I I I I I I

11 6. Mid premeiotic interphase I 3 4 Mean 7. Late premeiotic interphase-leptotene I 3 4 Mean 8. Zygotenepachy tene I Mean 9. First metaphase I Mean 10. Young rnicrospores I 3 Mean The estimated nuclear volume (n.v.), total centromere volume (~c.v.), and total nucleolar volume (znlr.~.), and the chromosome number (n) and (where known with certainty) the C value, and DNA content (DNA) of nuclei from ID tissueo or stages of Triticum aestivum cv. Chinese 1 Spring, together with derived estimates of the proportion of the total nuclear volume occupied by centromeres expressed as a percentage cb (&.v./n.v. %), the mean volume of centromeric material per pg of nuclear DNA (Zc.v./DNA). The number of sections per nucleus is (N.B. The asterisk in column indicates a nucleus stained with phosphotungstic acid.) & -

12 10 M. D. Bennett and others mean total volumes of CeSs per zc nucleus in these species (4-87 and 3-85 /tm 3 ) are similar whereas their respective mean centromere volumes per zc chromosome (0-33 and 0-17 /tm 3 ) are different. Clearly, therefore, total volume of CeSs per nucleus is largely characteristic of a particular mass of nuclear DNA and largely independent of the number of chromosomes between which that mass is distributed. However, the total volume of CeSs per nucleus can sometimes be directly proportional to chromosome number, for example, when zn increases due to polyploidy while the DNA content per constituent diploid genome remains constant (or almost so). Thus, the total volume of CeSs per nucleus is closely related to zn when Triticum dicoccum (zn = zx = 8) and T. aestivum (zn = 6x = 4) are compared (Table ) I I Centromere volume, (im J Fig. 5. The relationship between C DNA amount and the mean total volume of CeSs per zc chromosome complement in u higher plant species. The interspecific relationship between total centromere volume per nucleus and nuclear DNA amount Fig. 5 illustrates the highly significant relationship (r = 0-96; P < o-ooi) between the mean total volume of CeSs per nucleus and the zc DNA amount of 11 higher plant species. The computed linear regression line cuts the ordinate at a value not significantly different from zero. On average the 6 nuclei contained / tms f centromere per 1 pg of DNA, but values for individual nuclei differed by -8-fold ranging from 0-11 (in Hordeum vulgare) to 0-76 /6m 3 (in Triticum monococcum). Thus, variation in the volume of centromere material per 1 pg of DNA was small compared with the much larger ranges in total centromere volume per nucleus (4-fold) and DNA content per nucleus (i - 3-fold). In other words, the volume of

13 Nuclear DNA content and centromere volume 103 centromere material per pg of DNA was approximately constant despite the large interspecific variation in nuclear DNA content. Components of intraspecific variation in centromere volume Examination of the data in Table 3 suggests that variation in nuclear DNA content was also an important cause of intraspecific variation in the total volume of CeSs per nucleus. Thus, comparison of 10 nuclei with either known ic (3 young microspores), C (4 meiocytes at early premeiotic interphase), or \C (1 archespore and meiocytes) shows first a significant relationship (Fig. 6) between total volume of CeSs per nucleus and nuclear DNA content (P < o-ooi; r = 0-96), and secondly, the mean total volume of CeSs for the 3 classes of nuclei (-81, 6-85 and 1-94 /on 3, respectively) differ in the ratio 11-4:4-6, which is close to the known 1::4 rat ' of their DNA contents. 70 r- 60 s z Q _L Centromere volume, /im' Fig. 6. The relationship between nuclear DNA content and the total volume of CeSs per nucleus in 10 nuclei of Chinese Spring. Supporting evidence comes from comparing nuclei with C (early premeiotic interphase) or 4C (pachytene) in Triticum monococcum (Table ) where the total volume of CeSs per nucleus was almost exactly double in the latter (717-8 /*m 8 ) compared with the former (346-3 /mi 3 ). The slopes of the regression lines for nuclear DNA content on total volume of CeSs per nucleus in the intraspecific (b = 4-54) and interspecific (b = 4-89) comparisons are very similar (Fig. 7). Thus, the consequences of interspecific and intraspecific variation in chromosome DNA content on centromere volume are apparently virtually the same.

14 104 M. D. Bennett and others Further examination of the data in Table 3 shows that developmental variation independent of, and superimposed on, that attributable to differences in nuclear DNA content, was also a cause of intraspecific variation in the total volume of CeSs per nucleus in Chinese Spring. For example, although the DNA content of the sampled nuclei differed by 4-fold, the total volume of CeSs per nucleus differed by up to 7-fold. The total volume of CeSs in one root-tip nucleus (-67 /tm 3 ), whose minimum DNA content was C, was smaller than the corresponding value for a young microspore nucleus containing the ic DNA amount (-81 fim 3 ). Thus, there was at least a -i-fold range of variation in centromere volumes not attributable to variation in nuclear DNA content in Chinese Spring. /u 60 / Q. C s c Qo Q clea Z / / / / / / / / / / y / / A // // 10 - / / / / i i i i i i Centromere volume, jjm- 1 Fig. 7. A comparison of the regression lines for nuclear DNA content on total volume of CeSs per nucleus in the interspecific ( ) and intraspecific ( ) comparisons. The typical extent of intraspecific variation in centromere volume not attributable to differences in nuclear DNA content is unknown. However, it probably approximates to the extent of intraspecific variation in total chromosome volume per nucleus known to occur independent of variation in nuclear DNA content, i.e. about -4-fold in Vicia faba (Bennett, 1970) and Secale cereale (Bennett & Rees, 1969). If so, the present results are probably typical in showing'that variation in centromere volume not attributable to variation in nuclear DNA content (-1-fold) has an important, but minor, role in determining the overall intraspecific range of variation in centromere volume, while variation attributable to differences in nuclear DNA content (4-fold) plays the major role. The present variation in centromere volume which is not attributable to variation in nuclear DNA content presumably involves variation

15 Nuclear DNA content and centromere volume 105 in other constituent materials of chromosomes, notably, protein and RNA (Bennett & Rees, 1969; Bennett, 1970). The relationship between total centromere volume and nuclear volume As noted in the introduction, nuclear DNA content is closely correlated with nuclear volume in higher plants (Rees et al. 1966; Pegington & Rees, 1970). It is therefore reasonable to expect total centromere volume per nucleus to be positively correlated with nuclear volume, as both of these characters are similarly correlated with nuclear DNA content. This expectation is realized for both interspecific and intraspecific comparisons. For example, Fig. 8 illustrates the highly significant positive relationship (r = 0-95; P < o-ooi) between nuclear volume and the mean E a. o Centromere volume, nm 3 I 1 16 Fig. 8. The relationship between mean nuclear volume and the mean total volume of CeSs in 11 higher plant species. total volume of CeSs per nucleus for the 11 species listed in Table. Overall, in the 6 nuclei sampled, CeSs occupied % f tne nuclear volume, but values for individual nuclei showed a 6-3-fold range from % m a Vicia faba nucleus to -104% in a Briza maxima nucleus. The relationship between nuclear volume and centromere volume may differ between dicots and monocots. Thus, the mean percentage of the nuclear volume occupied by the centromeres in 3 dicot nuclei (0-407 %) was not quite half the corresponding value (0-835 %) f r 3 monocot nuclei. Although this difference is statistically significant (P < o-ooi), tests of its biological significance must await studies of larger samples of dicot nuclei and species than in the present work. However, it should be noted that large differences between monocots and dicots

16 io6 M. D. Bennett and others in nuclear characters including the density of DNA in metaphase chromosomes have been noted previously (Evans & Rees, 1971)- Values for the proportion of the nuclear volume occupied by CeSs in monocot nuclei showed only a 3-fold range from o-66% in Tradescantia paludosa to -10% in the larger Briza nucleus. The overall range of variation in the proportion of the nucleus occupied by CeSs (6-3-fold) was much smaller than either the 4-fold range in total volume of CeSs per nucleus or the i4-7-fold range in nuclear volume. A given volume of CeSs is apparently characteristic of a relatively narrow range of nuclear volumes, at least in monocots. 500 r 000 E a I I Centromere volume, Fig. 9. The relationship between nuclear volume and the total volumes of CeSs per nucleus in 9 nuclei of Chinese Spring. Fig. 9 illustrates the highly significant positive intraspecific relationship (r = 090; P = < o-ooi) between total volume of CeSs per nucleus and nuclear volume estimated in 9 nuclei from 10 different developmental stages in Chinese Spring. (N.B. The meiocytes at first metaphase are excluded because they lacked nuclear membranes, so that their nuclear volume could not be estimated.) Similar relationships were also found when 3 sub-groups of nuclei were compared: (1) nuclei from root-tip or anther connective cells (r = o-88; P = 0-0), () 6 germ line archesporial cells at premeiotic mitotic cycles (r = 0-97; P = o-ooi), and (3) 14 nuclei at premeiotic interphase or first meiotic prophase (r = o-8i; P < o-ooi). Overall, in the 9 nuclei, centromere material occupied 0-840% of the nuclear volume. However, values for individual nuclei showed about a --fold range from 0-59% in a root-tip nucleus, to 1-18% in a meiocyte near leptotene. The overall mean (0-840 %) is very similar to that for 6 monocot nuclei from 9 species (0-835 %)»

17 Nuclear DNA content and centromere volume 107 while the intraspecific range is close to that noted from the interspecific comparison of monocot species (i.e. o-66 to -10%). As in the interspecific comparison, variation in the proportion of the nuclear volume occupied by centromeres (--fold) is small compared with the 7-fold ranges in nuclear volume and total volume of CeSs per nucleus. Thus, the total volume of CeSs occupied a narrowly variable proportion of nuclear volume, despite the large intraspecific variation exhibited by the latter. Fig. 10 compares the regression lines for nuclear volume on total volume of CeSs per nucleus for the interspecific and intraspecific comparisons. The slopes of the lines (b = and 99-5, respectively) are significantly different (P < o-ooi) E a * 100 J Centromere volume, /jm 3 Fig. 10. A comparison of the regression lines for nuclear volume on total volume of CeSs per nucleus in the interspecific ( ) and intraspecific ( ) comparisons. Total centromere volume and nucleolar volume Nuclear DNA content is positively correlated with the total mass of nucleolar material per nucleus in interspecific comparisons of higher plants (Pegington & Rees, 1970; Paroda & Rees, 1971). Consequently, total nucleolar volume is also expected to be positively correlated with centromere volume, as explained in the previous section. Testing this expectation for the 11 species listed in Table shows that the relationship between mean total nucleolar volume per nucleus and mean total volume of CeSs per nucleus closely approaches significance (r = 0-58; P = ). One reason for this low correlation coefficient may be the fact that the nucleolar activity differs greatly in plants between first meiotic prophase (Moss & Heslop-Harrison, 1967) and premeiotic interphase. Results for the 3 species in which meiotic nuclei were sampled (Briza maxima, Festuca hybrid, and Tradescantia paludosa) do show

18 io8 M. D. Bennett and others a significant relationship (r = 0-99; P < o-ooi) between nucleolar volume and total volume of CeSs per nucleus, as do results for the remaining 8 species sampled at early premeiotic interphase (r = 0-7; P < 0-05). The distribution of points for Crepis capillaris and Viciafaba in Fig. 11 may indicate another potential difference between dicots and monocots E a Centromere volume, y.m y Fig. 11. The relationship between mean nucleolar volume per zc nucleus and mean total centromere volume per nucleus in dicots (O) and 6 monocots ( ) at premeiotic interphase, and in 3 monocots at first prophase of meiosis (A)- Values plotted for the latter monocots are half the estimates for 4C nuclei given in Table. An intraspecific comparison of total nucleolar volume per nucleus and mean total volume of CeSs reveals results similar to those just noted for the interspecific comparison. Thus, there is a highly significant positive relationship between these characters (r = 077; P < o-ooi) for a sample of 9 nuclei at 9 developmental stages in Chinese Spring (Fig. 1). (N.B. The nuclei at first metaphase are omitted from this comparison as they lack a nucleolus.) However, the correlation coefficient is again lower than for the intraspecific relationships between centromere volume and both nuclear DNA content (r = 0-96) and nuclear volume (r = 0-90). The low overall correlation between nucleolar volume with total volume of CeSs per nucleus is reflected in tests for 3 sub-samples: (1) 6 somatic nuclei (r = 0-15; P = 0-784), () 6 archesporial cells (r = 0-77; P = 0-073), ar >d (3) meiocytes (r = 0-48; P = 0-080). The nucleolus often forms slowly after the nuclear membrane and is dispersed before the nuclear membrane at nuclear division, which may largely explain

19 Nuclear DNA content and centromere volume 109 why nucleolar volume in both the interspecific and intraspecific comparisons was less correlated with total volume of CeSs per nucleus than nuclear DNA content and nuclear volume E a I I I Centromere volume, Fig. 1. The relationship between nucleolar volume and the total volume of CeSs per nucleus in 9 nuclei of Chinese Spring. DISCUSSION The extent and nature of variation in centromere volume The present results for Triticum aestivum agree with those for Allium fistulosum (Church & Moens, 1976; Moens & Church, 1977) in showing considerable intraspecific variation in the total volume of CeSs per nucleus (about 7-fold in the former, compared with about -7-fold in the latter). The present work also reveals even greater variation in this character for interspecific comparisons (about 4-fold). Despite this large variation in the total volume of CeSs per nucleus, both within and between higher plant species, the appearance of centromeric material remained very similar. Clearly therefore, the total volume and mass of centromeric material per nucleus are very variable. However, the present work shows that such variation is not random, but occurs within narrow limits related to the nuclear environment. Thus, the total volume of CeSs per nucleus shows remarkably close positive correlations with nuclear DNA content (Figs. 5, 6) and nuclear volume (Figs. 8, 9), and to a lesser extent with total nucleolar volume per nucleus, for both intra- and interspecific comparisons. There can be little doubt that the relationship with nuclear DNA content is of primary importance, not only because nuclear DNA content consistently displayed the highest correlation coefficients with total volume of CeSs per nucleus, but also because of its causal role in determining phenotypic characters. The present results also clearly establish that centromere volume can also reflect developmental variation in nuclear volume independent of nuclear DNA content

20 no M. D. Bennett and others which involves developmental variation in the other major components of chromatin (protein and RNA). However, such developmental fluctuations in centromere volume are of minor amplitude compared with those associated with variation in nuclear DNA content. Taken together, the present results suggest that in general a given total volume of centromeric material is characteristic of an approximately constant nuclear volume and mass of nuclear DNA, but largely independent of the number of chromosomes per diploid nucleus. Moreover, the present results, together with those for a Festuca hybrid in the accompanying paper (Jenkins & Bennett, 1980), show that a centromere is not a unit structure of constant volume and mass. Rather it appears to be a chromosome segment whose highly variable volume closely reflects the size and mass of the chromosome to which it belongs. The DNA content of centromeres in relation to nuclear DNA content One reason for undertaking the present work was to try and assess whether large interspecific differences in DNA C-value sometimes involve proportional changes in the total volume and DNA content of small segments present on every chromosome, using centromeres as small defined test segments. Measurements of the DNA contents of centromeres are not available in any species, and therefore a direct comparison of the DNA contents of nuclei and their centromeres cannot be made. However, in Chinese Spring the total volume of CeSs per nucleus varied almost directly in proportion with developmental differences in total nuclear DNA content (Fig. 6), and hence presumably with the total DNA content of CeSs. The total volume per nucleus of CeSs of constant appearance also increased almost directly in proportion with nuclear DNA amount for the interspecific comparison of n species with an 8-8-fold range of C DNA values (Fig. 5). If, as seems most likely, the density of DNA in the centromeric material of these species was approximately constant, the large interspecific variation in C-value must have included approximately corresponding variation in the total DNA content of CeSs per nucleus. Thus the present results for centromeres provide probably the first example to indicate that variation in the total DNA content of small segments present on each chromosome sometimes varies directly in proportion to large interspecific variation in nuclear DNA C-value. Centromere volume in the context of the nucleotype The present correlations between centromere volume and several other nuclear characters are probably a small part of the syndrome of proportional variation involving a complex of characters, including nuclear DNA content, whose widely investigated interrelations (Bennett, 1973; Underbrink & Pond, 1976; Cavalier-Smith, 1978) are largely nucleotypic in origin. Nucleotypic variation describes those phenotypic characters determined as the consequences of variation in the mass and volume of nuclear DNA but independent of its encoded information (Bennett, 1971, 197). The nucleotype is therefore a genotypic but not a genie character. The well known correlation between total chromosome volume of CeSs and nuclear DNA content (Fig. 5) apparently describes a part of this general nucleotypic correla-

21 Nuclear DNA content and centromere volume 111 tion as it affects a small, functionally distinct fraction of the genome, represented by a small segment present on each chromosome. Speculation regarding the cause of the present correlations involving centromere volume would be premature until more is known about the functional significance of variation in centromere size, and the mechanism which controls it. The functional significance of variation in centromere volume The highly significant relationships between total centromere volume per nucleus, and nuclear size and DNA content, are presumably of some functional significance, although their nature is unknown. The number of spindle microtubules attached to each centromere is normally greatly in excess of the number required to produce the force needed to move the chromosome at karyokinesis (Forer, 1969). Consequently, it is difficult to envisage the present relationship between centromere volume and chromosome mass as a device for ensuring that each chromosome competing for spindle microtubules obtains enough for efficient karyokinesis, unless perhaps this is important only at a particular stage in the life cycle or at rare times of severe physiological stress. Alternatively, the control of centromere volume, within narrow limits, may be determined by some other function(s) of the centromere unrelated to the force required to move chromosomes. For example, it has been suggested that centromeres play an important role in establishing and/or maintaining the coorientation of chromosomes; in particular, the somatic association of homologues and homoeologues (Feldman, Mello-Sampayo & Sears, 1966). If so, the control of centromere volume close to an optimum proportion of chromosome and nuclear size may relate to the mechanical needs of ensuring that sufficient spindle microtubules attach to each chromosome rigorously to maintain a particular spatial ordering of chromosomes throughout karyokinesis. The determination of centromere volume The present results clearly indicate that some mechanism determines that the volume and probably the DNA content of centromeres will reflect closely the DNA content of the chromosomes containing them. This mechanism is obscure. However, it will be important to discover whether it is a positive control (perhaps like that which adjusts the number of ribosomal RNA genes at the Bobbed locus in Drosophila (Ritossa, 1976)), or the result of natural selection. It seems pertinent to pose questions. First, over what interval does the mechanism determining centromere volume act? Second, what are the consequences if the ratio of centromere volume to DNA content for a chromosome suddenly deviates from the normally narrow range of values? Both of these questions should be answered by simple experiments. For example, comparing the relative centromere volume of a known chromosome before, and at intervals after, the loss of a large non-centromeric segment should show whether the reduction in relative chromosome size and DNA content is reflected by a similar reduction in relative centromere volume, or whether the centromere retains its original volume characteristic of the undeleted chromosome.

22 ii M. D. Bennett and others The former result would indicate that centromere volume is subject to a short-term control by a mechanism which monitors centromere volume on a chromosomal basis. The latter result would indicate that centromere volume is probably determined by natural selection. It would also illustrate the consequences of a sudden increase in the ratio of the volume of the centromere in relation to chromosome volume. Thus, if such a chromosome were transmitted normally within and between generations this would indicate that, although the ratio of centromere volume to chromosome size is eventually determined within narrow limits, nevertheless, in the short term, considerable variation around these norms is tolerated and its consequences are neither immediate nor serious. Such studies will have a particular interest because large deviations from the normal relationship between relative centromere volume and relative chromosome size and DNA content could be a cause of nuclear instability and the selective elimination of single chromosomes or even whole genomes. An intranuclear correlation of centromere volume and chromosome DNA content and its potential uses? As the total volume of CeSs per nucleus is closely correlated with nuclear DNA content (Figs. 5, 6), it is interesting to question whether the volumes of individual centromeres within a nucleus are also correlated with the DNA contents of the chromosomes to which they belong, and if so, whether the correlation is close enough to allow unequivocal identification of a particular centromere in a single nucleus using estimates of relative centromere volume alone. Unfortunately estimates are not available for any species for both the relative DNA contents of each chromosome and of the volume of each centromere, so a direct test must await further work. However, relative chromosome DNA content and length are frequently almost directly proportional in higher plants (Barlow & Vosa, 1969; Heneen & Casperson, 1973), and measurements of relative chromosome lengths are available for many species. Thus, a comparison of the relative volumes of single centromeres with relative lengths of individual chromosomes, while of interest in its own right, should also indicate whether the volumes of single centromeres are correlated with the relative DNA contents of single chromosomes within a nucleus. If centromere volume is directly proportional to chromosome length within a species then the range of volumes of single centromeres should correspond with the range of individual chromosome lengths, and/or DNA contents. Meaningful estimates of the range of volumes of single centromeres are available for 3 higher plant species. Each shows a close correspondence between the ratio of the volumes of the smallest to the largest centromeres and either the ratio of the lengths of the shortest to the longest chromosome, or the ratio of the smallest to the largest DNA contents of single chromosomes (Table 4). It was decided, therefore, to make a preliminary investigation of the intranuclear relationship between the volumes of single centromeres and the lengths and DNA contents of single chromosomes, and a Festuca hybrid was chosen as a suitable material because of its large range of chromosome DNA contents. Although the DNA contents of individual chromosomes in either of the parents (F. scariosa and F. drymeja) differed by less than i-5-fold, the range in the hybrid is

23 Nuclear DNA content and centromere volume 113 much greater because the parent species, while having the same chromosome number (zn = x = 14), differ by 36% in their DNA C-values. The results of this preliminary study, presented in the accompanying paper (Jenkins & Bennett, 1980), show: first, that the observed range of volumes of single centromeres almost exactly matched the range of single chromosome DNA contents (both about -3 - Table 4). Secondly, they show that the frequency distribution of observed centromere volumes was not significantly different from the frequency distribution predicted, assuming direct proportionality between centromere volume and chromosome length and DNA content. This indicates that there is a positive relationship between centromere volume and chromosome size. It was concluded, therefore, that the relationship between centromere volume and nuclear volume and DNA content noted for whole nuclei in the present work probably also applies to Table 4. Comparison of the range of centromere volumes with the ranges of chromosome lengths and or chromosome DNA contents in 4 higher plants taxa Species Allium fistulosum Festuca scariosa x drymeja Triticum monococcum Triticum acstivum Ratio of vol. of largest to smallest centromeres, approx. 1:-3* 1:1-3* I:I-5 5 Relative lengths of longest and shortest chromosomes, approx. 1:1-5' Relative DNA contents of biggest and smallest chromosomes, approx. 1:-3* 1:1-4' Key to references: 'Church & Moens (1976); 'Vosa (1976); "Jones & Rees (1968); 'Jenkins & Bennett (1980); 'Bennett (unpublished data); 'Giorgi & Bozzini (1969); 'Nishikawa (1970). chromosomes within a nucleus of the Festuca hybrid. The extent of this intranuclear correlation is unknown, but it seems reasonable to expect that it will be of general significance. Although it was not possible to identify unequivocally any single centromere in any single nucleus of the Festuca hybrid, it is probably possible to identify the centromere belonging to the largest chromosomes in the haploid complement of Allium fistulosum (Jenkins & Bennett, 1980). Assuming that the results for these materials are typical, then the correlation between centromere volume and chromosome length and DNA content is probably close enough, and the present techniques used to estimate centromere volume are probably accurate enough, to allow the unequivocal identification of centromeres in single nuclei of some suitable materials. Thus, the way is probably open for direct observations of the intranuclear disposition of centromeres of small marker chromosomes (such as B-chromosomes and some telocentrics) and of one or more A-chromosomes in materials where the difference in DNA content or length between chromosomes is large. If so, it is anticipated that such studies should permit valuable reinvestigations of several aspects of the spatial distribution of chromosomes using, for the first time, interphase and dividing nuclei undistorted by squashing. Such aspects which merit attention include: (1) somatic

24 ii4 M. D. Bennett and others association of homologues and homoeologues (Feldman & Avivi, 1973); () premeiotic alignment of homologues (Dover & Riley, 1977); (3) secondary association of homoeologues (Kempanna & Riley, 1964); and (4) ordered end-to-end associations of heterologues (Comings, 1968; Ashley, 1979). REFERENCES ASHLEY, T. (1979). Specific end-to-end attachment of chromosomes in Ornithogalum virens. J. Cell Sci. 38, BARLOW, P. W. (1977). Determinants of nuclear chromatin structure in Angiosperms. Annls Set. not. Bot. Biol. vigit., Ser. 1, 18, BARLOW, P. W. & VOSA, C. G. (1969). The chromosomes of Puschkinia libanotica during mitosis. Chromosoma 7, BEDBROOK, J. R., JONES, J., O'DELL, M., THOMPSON, R. D., FLAVELL, R. B. (1980). A molecular description of telomeric heterochromatin in Secale species. Cell 19, BENNETT, M. D. (1970). Natural variation in nuclear characters of meristems of Vicia faba. Chromosoma 9, BENNETT, M. D. (1971). The duration of meiosis. Proc. R. Soc. B 178, BENNETT, M. D. (197). Nuclear DNA content and minimum generation time in herbaceous plants. Proc. R. Soc. B 181, BENNETT, M. D. (1973). Nuclear characters in plants. Brookhaven Symp. Biol. 5, BENNETT, M. D., GUSTAFSON, J. P. & SMITH, J. B. (1977). Variation in nuclear DNA in the genus Secale. Chromosoma 61, BENNETT, M. D., RAO, M. K., SMITH, J. B. & BAYLISS, M. W. (1973). Cell development in the anther, the ovule, and the young seed of Triticum aeslivum L. var. Chinese Spring. Phil. Tram. R. Soc. B 66, BENNETT, M. D. & REES, H. (1969). Induced and developmental variation in chromosomes of meristematic cells. Chromosoma 7, BENNETT, M. D. & SMITH, J. B. (1976). Nuclear DNA amounts in angiosperms. Phil. Trans. R. Soc. B 74, BENNETT, M. D., SMITH, J. B., SIMPSON, S. & WELLS, B. (1979). Intranuclear fibrillar material in cereal pollen mother cells. Chromosoma 71, CAVALIER-SMITH, T. (1978). Nuclear volume control by nucleoskeletal DNA, selection for cell volume and cell growth rate, and the solution of the DNA C-value paradox, jf. Cell Set CHURCH, K. & MOENS, P. B. (1976). Centromere behaviour during interphase and meiotic prophase in Alliumfistulosumfrom 3-D, EM reconstruction. Chromosoma 56, COMINGS, D. E. (1968). The rationale for an ordered arrangement of chromatin in the interphase nucleus. Am. J. hum. Genet. 0, DOVER, G. A. & RILEY, R. (1977). Inferences from genetical evidence on the course of meiotic chromosome pairing in plants. Phil. Trans. R. Soc. B 77, EVANS, G. M. & REES, H. (1971). Mitotic cycles in dicotyledons and monocotyledons. Nature, Lond. 33, 35O-35I- FELDMAN, M. & AVIVI, L. (1973). The pattern of chromosomal arrangement in nuclei of common wheat and its genetic control. Proc. 4J/1 int. Wheat Genet. Symp. Columbia, U.S.A., pp FELDMAN, M., MELLO-SAMPAYO, T. & SEARS, E. R. (1966). Somatic association in Triticum aestivum. Proc. natn. Acad. Set. U.S.A. 56, FORER, A. (1969). Chromosome movements during cell-division. In Handbook of Molecular Cytology (ed. A. Lima-de-Faria), pp Amsterdam: North-Holland. GILLIES, C. B. (1973). Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes. Chromosoma 43, GIORGI, N. & BOZZINI, A. (1969). Karyotype analysis in Triticum Ill-Analysis of the presumed diploid progenitors of polyploid wheats. Caryologia, HENEEN, W. K. & CASPERSON, T. (1973). Identification of the chromosomes of rye by distribution patterns of DNA. Hereditas 74, 59 7.

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