A. D. KIDD*, D. FRANCISf and M. D. BENNETTJ

Annals of Botany 59, 603-609, 197 603 Replicon Size, Mean Rate of DNA Replication and the Duration of the Cell Cycle and its Component Phases in Eight Monocotyledonous Species of Contrasting DNA C Values A. D. KIDD*, D. FRANCISf and M. D. BENNETTJ 'Department of Agriculture, University of Reading, Whiteknights, Reading, RG6 H, UK; ^Department of Plant Science, University College, P.O. Box 7. Cardiff, CF1 1XL, UK; and \ Plant Breeding Institute, Moris Lane, Trumpington, Cambridge, CB LQ, UK Accepted: II November 196 ABSTRACT Various aspects of the cell cycle were measured in the apical meristem of primary and seminal roots of eight monocotyledonous angiosperms: Oryza saliva (0-6 pg), Zea mays (-4 pg), Pennisetum americanum (-5 pg), Aegilops umbellulala (51 pg), Hordeum vulgare (5-5 pg), Triticum monococcum (6- pg), Secale cereale (-6 pg) and Tulipa kaufmanniana (-6 pg), representing a 3-fold variation in DNA C values. Using 4-dold roots of the first seven species and 1-d-old Tulipa roots, replicon size and rates of replication were determined by DNA fibre autoradiography, and the duration of the cell cycle and its component phases by the percentage labelled mitoses method. When tested with DNA C value, no significant relationships existed for replicon size, rate of DNA replication or duration of G1. Significant positive linear relationships were found between DNA C vajue and cell cycle duration, duration of mitosis and G duration when all data were tested, but not when the Tulipa data were excluded. The only characters significantly related to DNA C value when the Tulipa data were included or excluded were the duration of S-phase, and the ratio of the interval required for a replicon to replicate its allotted DNA (Rs) to the duration of S-phase (Ds). The Rs:Ds ratio is a measure of synchrony of replicon activation, and the higher the DNA C value the lower this ratio became. We concluded that there was a nucleotypic effect of DNA C value on this ratio and that the interval between activation of replicons became protracted and hence S-phase lengthened as C value increased. Key words: Cell cycle, DNA C value, DNA replication, replicon, S-phase. INTRODUCTION In angiosperms, there is at least a 500-fold variation in DNA C values (Bennett, 195), where one Cis the amount of DNA in the unreplicated nuclear genome of a gamete. Despite large interspecific increases in C values both within a genus, and even within major groups of eukaryotes, there is often Little variation in the amount, type and control of gene expression. The major contribution to increase in DNA C value is non genie, repetitive DNA sequences which can account for greater than 90 per cent of the total nuclear DNA (Flavell, 190). However, large changes in nuclear genome size are far from neutral in their effects on organisms. For example, there is a strong positive correlation between nuclear DNA mass (defined by Bennett, 197, as the nucleotype) and the 03O5-7364/7/O6O603 + O7 $03.00/0 duration of the mitotic cell cycle in the root meristem in a range of diploid monocots (Van't Hof and Sparrow, 1963; Bennett, 197). A similar positive correlation exists between DNA C value and the duration of meiosis (Bennett, 1977). These so-called nucleotypic effects are not confined to the cellular level but extend to whole plant characters such as geographical distribution and minimum generation time (minimum time from germination to first production of mature seed) (Bennett, 197, 1976). The question remains as to how these nucleotypic effects are determined and linked. The rate at which nuclear DNA is doubled during DNA synthetic-(s)-phase of the cell cycle may be partly responsible since the greater the amount of DNA, the longer the interval required to replicate it 197 Annals of Botany Company

604 Kidd et al. DNA Replication and DNA C Values (Van't Hof, 1965). Van't Hof and Bjerknes (191) measured rates of DNA replication and replicon size in root meristematic cells of seven diploid dicot angiosperms, one tetraploid dicot and one diploid monocot, with genome sizes which differed -fold and whose S-phase durations differed fourfold. These species had similar replicon sizes ranging between 16 and 7/tm and replication rates, per single replication fork between 6 and 10 fim h" 1. The conclusion was that the duration of S-phase was determined by a minimal number of replicons that function sequentially during DNA replication. In other words, as C value increases the minimal replicon number increases and S-phase gets longer. Such analyses often draw on published data on S-phase duration to augment primary data on rates of DNA replication (see Van't Hof and Bjerknes, 191; Francis, Kidd and Bennett, 195). Also it is difficult to make meaningful conclusions about the pattern of replicon activation in relation to variation in DNA C value when diploids are compared with auto and allo-polyploids. To obviate these problems we have measured rates of DNA replication, replicon size, the duration of the cell cycle and its component phases in the apical 1 mm of primary and seminal roots at similar stages of development in eight diploid monocots at 0 C. The DNA C values of these species ranged from 0-6 pg for Oryza sativa to -6 pg for Tulipa kaufmanniana (Bennett and Smith, 1976). Thus, the present work describes the pattern of replicon activation and the cell cycle in species of identical ploidy but which were specifically chosen to provide a near 40-fold variation in DNA C values. MATERIALS AND METHODS Seeds of Zea mays cv. Seneca 60 (/7 = x = 0), Pennisetum americanum (/J = x = ), Aegilops umbellulata (/J = Ix = ), Hordeum vulgare cv. Sultan (/7 = x = ), Triticum monococcum (/7 = x = ) and Secale cereale cv. Dominant (/i = x = ) were surface-sterilized in per cent (v/ v) sodium hypochlorite solution, rinsed in sterile, distilled water, imbibed in Petri dishes and grown on aseptically at 0 C in darkness for 4 d. Seeds of Oryza sativa cv. IR 34 (/7 = x = 4) were germinated aseptically at 30 C and grown in darkness for 3 d before transfer to 0 C for one day. Bulbs of Tulipa kaufmanniana cv. The Kaufmanniana (n = x = 4), supplied by J. Amand, Beethoven Street, London, were surface-sterilized as above, grown in vermiculite for 1 days at 0 C in darkness. Fibre autoradiography Four-d-old primary and seminal root tips of O. sativa, Z. mays, P. americanum, A. umbellulata, H. vulgare, Triticum monococcum and Secale cereale and 1-d-old root tips of Tulipa kaufmanniana were exposed at 0 C, to high specific activity (-6-31 TBq mmol" 1 tritiated-(methyl-*h)-thymidine (*H-TdR) at a concentration of 37 mbq ml" 1 for various lengths of time. Nuclei were isolated and lysed on subbed microscope slides and the DNA was spread (Van't Hof and Bjerknes, 1977). The slides were dipped in Ilford K, photographic emulsion at 40 C, air-dried at 5 C at a relative humidity of 40 to 50 per cent (Rogers, 1979) and subsequently left for three to four months at 5 C C in the presence of a desiccant. They were developed using Ilford Phenisol and prepared as permanent autoradiographs. Rates of DNA replication per single replication fork, referred to henceforth as replication fork movement, and replicon size were determined based upon published methods (Van't Hof and Bjerknes, 1977; Francis and Bennett, 19). Cell cycle measurements The roots of 4-d-old seedlings and 1-d-old bulbs were suspended in s H-TdR for 0-5 h (concentration 37/iBq ml" 1 ; specific activity 15 GBq mmol" 1 ) followed by a cold chase with lo" 6 *! TdR for 0-5 h before being grown on in sterile distilled water at 0 C in darkness. Roots were fixed in 1:3 (v/v) glacial acetic acid: absolute alcohol at 1- h intervals following exposure to H-TdR. They were hydrolysed in 1N HO for -10 min at 60 C or in 5 N HC1 for 30 min at 5 C and stained by the Feulgen reaction. Squash preparations were made of the apical 1 mm of each root tip, permanent autoradiographs were prepared as before and the percentage of labelled mitoses (PLM) (Quastler and Sherman, 1959) was determined for three slides prepared from three root tips fixed at each sampling time. Statistical analyses The relationship between the various characters measured was examined by regression analysis. However, because the DNA C value of Tulipa kaufmanniana (-6 pg) is three times greater than that of the next highest C value (Secale cereale -6 pg) the C value data exhibit a marked discontinuity along the X-axis and thus do not conform to the bivariate distribution required by linear regression (Sokal and Rohlf, 191). Accordingly, separate

Kidd et al. DNA Replication and DNA C Values 605 0 7 A E 3- a Icon a. if 1 16 1 3 * 5 4* 6 «10 45 60 90 Exposure time (mln) 10 FIG. 1. The relationship between mean (± s.e.) replicon size (/un) and duration of *H-TdR pulse (min) for 4-dold root meristems of Oryza saliva L. at 0 C. Extrapolation of the regression to the y-axis gave replicon size. linear regressions were performed including or excluding Tulipa data. In addition a non-parametric regression procedure, the Spearman-Rank correlation, which has no prerequisite for particular data distribution was used. In all cases the critical level of significance was taken as P = 005. Replicon size RESULTS AND DISCUSSION Replicon size was estimated by the mid-point to mid-point-(mm)-method of Van't Hof and Bjerknes (1977). An MM measurement comprises the distance from the gap between one tandem array of silver grains to the corresponding gap of an adjacent tandem array. These measurements tend to peak at a particular modal class size representing an average replicon size for that species (e.g. 0-5 fan for Secale cereale cv. Petkus Spring (Francis and Bennett, 19)). Moreover the shorter the exposure time the stronger modal replicon size becomes (Van't Hof, Bjerknes and Clinton, 1979). In the present study, rather than using class-size or mean MM values obtained from a given exposure time (Van't Hof and Bjerknes, 1977), we have determined replicon size from the relationship between mean size calculated from individual MM measurements, and exposure time (Fig. 1). Extrapolation of the regression to the >>-axis gave a value for replicon size for each species which was subsequently plotted against DNA C value (Fig. A). 1 0 repllc 0 Rate 6 4 6 4 * 5 7 4 6 10 DNA C value (pg) FIG.. The relationship between A, extrapolated mean replicon size (jim) and DNA C value and B, mean rate of DNA replication per single replication fork Omh" 1 ) and DNA C value for cells in the root meristem of eight diploid monocots at 0 C. Key: 1, Oryza saliva;, Zea mays; 3, Pemisetum americanum; 4, Aegilops umbellulata; 5, Hordeum vulgare; 6, Trilicum monococcwn; 7, Secale cereale;, Tulipa kaufmanniana. No significant relationship was established between our estimates of replicon size and DNA C value using either a linear regression of Spearman- Rank analysis (r = -0-44; rs = 001; />>(M)5). Van't Hof et al. (1979) suggested that in eukaryotes replicon size is conserved between 0 and 40 ftm. In the analysis of diploid and tetraploid angiosperms of Van't Hof and Bjerknes (191; see Introduction), mean replicon size was - ±0-4 fim, a value clearly within the limits of this notional conserved replicon. However, mean replicon size for the eight diploid monocots in the present study of 151 ± 1- ftm is clearly much smaller than that reported by Van't Hof and Bjerknes (191). This difference cannot be accounted for by the different method used here to estimate replicon size compared with that used by Van't Hof and Bjerknes (A. D. Kidd, upublished data). Although replicon size in Van't HoPs studies was normally measured at 3 C compared with 0 C in ours, such B

606 Kidd et al. DNA Replication and DNA C Values Q 30 0 1-10 30-0 E Q 1-0 A B «3» 5«6. 4» 4 6 10 ONA C value (pg) FIG. 3. The relationship between A, the duration (h) of the cell cycle (Dc) and DNA C value and B, the duration (h) of mitosis (Dm) and DNA C value for cells in the root meristem of eight diploid monocots at 0 C (for Key see Fig. ). temperature differences do not alter replicon size (Van't Hof, Bjerknes and Clinton, 197; A. D. Kidd and D. Francis, unpublished data). Thus it seems unlikely that replicon size is conserved in eukaryotes because its limits would need to be extended to include both the data presented here (Fig. A) and the measurements of 5/*m for Triticum aestivum cv Chinese Spring and allohexaploid triticale T7 (Francis et al, 195). 7» differences between our mean value and that of Van't Hof and Bjerknes (191). No significant relationship could be established between rate of DNA replication and DNA C value with or without the Tulipa data (r = 0-1 to + 0.47; rs = 0.05 to 006; P > 0-05; Fig. B;). These data are consistent with an earlier conclusion, based on fewer results, that close relationships between rates of DNA replication and DNA C value seem unlikely (Francis et al., 195). Duration of the cell cycle and its component phases A positive relationship was established between the duration of the cell cycle (Dc) in cells of the root meristem and DNA C value for all eight species (Fig. 3A; r = 0-94; P < 0001). However, all but one of the cell cycle values fall between approx 10 and h and it is only the very long cell cycle (30 h) and high C value (-6 pg) for Tulipa kaufmanniana which causes this regression to be significant. A corresponding regression analysis for the other seven species did not yield a significant linear relationship (r = 0-35; P > 0-05). Moreover, the corresponding Spearman-Rank correlations were non-significant (rs = 0-37 to 0-5; P > 005). This was also the case when the relationship between the duration of mitosis (Dm) and DNA C value was tested. The linear regression for all eight species was significant (r = 0S6;P = 0-01-0-0), but omitting the datum point for Tulipa resulted in a non-significant regression (r = 0-54; Fig. 3B; P > 0-05). The Spearman-Rank tests also resulted Rates of replication fork movement Mean rate of replication for the eight species was 4-6±0-6/*m h" 1 at 0 C, a rate about half that of the corresponding mean of 0 ± 0-6 /tm h "' obtained by Van't Hof and Bjerknes (191) for eight species grown at 3 C. A change in temperature from 0 to 3 C has been shown to affect rates of replication (Van't Hof et al, 197) so it is difficult to draw meaningful conclusions about the 4 6 10 ONA C value (pg) Fio. 4. The relationship between duration (h) of S-phase (Ds) and DNA C value in the root meristem of eight diploid monocots at 0 C (for Key see Fig. ).

Kidd et al. DNA Replication and DNA C Values 607 4 A 1 5 0-6.c 5 o 3 B 6 CN4 o 4 i # 3 6 4 7 4 6 10" DNA C vdue (pg) FIG. 5. The relationship between A, the duration (h) of Gl (DG1) and DNA C value and B, the duration of G (DG) and DNA C value for cells in the root meristem of eight diploid monocots at 0 C (for Key see Fig. ). in non-significant correlation coefficients (P > 0-05). Thus our results do not confirm earlier demonstrations of a nucleotypic effect of DNA C value on cell cycle duration in diploid monocots although the trend in the present work is clearly similar to that reported previously for a larger sample of 31 (Bennett, 197) compared with eight here, and for a smaller sample of eight monocot species (Evans and Rees, 1971). In contrast, a positive linear relationship was established between the duration of S-phase (Ds) and DNA C value whether the datum point for Tulipa was included (r = 0.9; P < O001) or excluded (r = O-3; P = 0-01-0-0). However, the Spearman-Rank test only gave a significant correlation coefficient when Tulipa was included (rs = 0-77; P = 0-0-0-05) but not when it was excluded (rs = 0-63; P>0-05; Fig. 4). These results are 4 6 10 ONAC value (pg) FIG. 6. The relationship between Rs:Ds ratio and DNA C value for cells in the root meristem of eight diploid monocots at 0 C (for Key see Fig. ). entirely consistent with a corresponding effect of DNA C value on S-phase duration in dicots (Van't Hof, 1965). Significantrelationshipswere not found between the duration of G 1 and C value (r = 0-; rs = -0-3; P 0O5; Fig. 5 A) nor between the duration of G and C value when the Tulipa data were excluded (r=-o5; rs=-0-; P>0-05; Fig. 5B). However the linear regression for all the G data was marginally significant (r = 0-75; P = 0-0 -0-05). Replicon synthesis in relation to S-phase The only component of the cell cycle which lengthened significantly in response to increasing DNA C value was S-phase. We used the S-phase data in combination with replicon size and replication fork movement data to estimate the time taken for a replicon to replicate its allotted DNA (Rs) in relation to the duration of S-phase (Ds) for TABLE 1. Chromosome number per genome, DNA C value (pg) and the time taken for a replicon to replicate its allotted DNA (Rs) in the root meristems of eight diploid monocots at 0 C Chromosome* number (n) C value (Pg) Rs (h) 1. Oryza saliva cv. IR34. Zea mays cv. Seneca 60 3. Pennisetum americanum 4. Aegilops umbellulata 5. Hordeum vulgare cv. Sultan 6. Trilicum monococcum 7. Secale cereale cv. Dominant. Tulipa kaufmanniana 4 0 4 06-4 -5 51 5-5 6- -6f -6 1-5 -4 1 1.6-1-6 1-4 1-5 * Bennett and Smith (1976) f Kidd unpublished data.

60 Kidd et al. DNA Replication and DNA C Values each species. Rs is calculated by dividing average replicon size by twice the rate of replication fork movement (Van't Hof et al., 197). For the eight diploid monocots used in this study Rs ranged from 1-4 to -4 h (Table 1). Thus, despite a 3-fold variation in DNA C values and the wide ranging values obtained for replicon size (Fig. A) and rates of replication fork movement (Fig. B), the time required for an individual replicon to replicate its allotted DNA was remarkably constant. Similarly Rs values in Van't Hof and Bjerknes (191) study ranged between 0-9 and -0 h. The latter had a somewhat lower limit than in the present study but this is largely predictable in view of their use of a higher temperature of 3 C compared with 0 C in our study. In eukaryotes replicons are organized into replicon families where its members are activated simultaneously at a specific time in S-phase (Van't Hof, 195). In effect this means that the Rs:Ds ratio is an indication of the degree of synchrony of replicon family activation and we have previously argued that the lower the ratio the more asynchronous replicon family activation becomes (see Francis et al., 195). Linear regression analysis revealed a significant negative correlation between Rs: Ds ratio and DNA C value whether the datum point for Tulipa was included (r = 0-91) or excluded(r= - 0-9 \;P = 0-01-0O; Fig. 6). Moreover, the Spearman-Rank test resulted in significant correlation coefficients both for all data (rs = -0-9; P = 0-001-0-01) and when the datum point for Tulipa was excluded (rs = 0-7; P = 0-0 -0-05). The causal nature of this relationship is unknown, but the existence of a significant relationship between nuclear genome size and synchrony of activation ofrepliconsstrongly suggests that there may be a nucleotypic effect of DNA C value on the Rs:Ds ratio. In other words, the higher the DNA C value the lower the Rs: Ds ratio and hence the more asynchronous activation of replicons becomes. This confirms thefindingsof Francis et al. (195) especially as in the present work we have measured replicons, rates of replication and Ds for the root meristem of each species under identical environmental conditions. Although Rs is fairly constant despite variations in DNA C value the significant decrease in Rs: Ds ratios indicates that as DNA C value increases the interval between the activation of clusters of replicons becomes protracted and hence S-phase gets longer. Perhaps the increase in the amount of repetitive sequences per basic genome, a common feature of increases in DNA C value (Flavell et al., 1974; Nagl, 19) is particularly related to asynchrony of replicon activation. ACKNOWLEDGEMENTS We thank the Agricultural and Food Research Council for support through Grant AG 7/4, and Professor D. B. Walden, Department of Plant Sciences, University of Western Ontario, Canada for supplying Zea mays cv. Seneca 60. LITERATURE CITED BENNETT, M. D., 197. Nuclear DNA content and minimum generation time in herbaceous plants. Proceedings of the Royal Society of London, Series B 11, 109-35. 1976. DNA amount, latitude and crop plant distribution. Environmental and Experimental Botany 16, 93-10. 1977. The time and duration of meiosis. Philosophical Transactions of the Royal Society of London, Series B 77, 01-06. 195. Intraspecific variation in DNA amount and the nucleotypic dimension in plant genetics, pp. 3-30. In Plant Genetics ed. M. Freeling. Alan R. liss, New York. and SMITH, J. B., 1976. Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London, Series B T74, 7-74. EVANS, G. H. and REES, H., 1971. Mitotic cycles in dicotyledons and monocotyledons. Nature 33, 350-1. FLAVELL, R., 190. The molecular characterisation of plant chromosomal DNA sequences. 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