Underreplication of repetitive DNA in polyploid cells of Pisum sativum

Experimental Ceil Research 98 (1976) 2 IO-22 1

UNDERREPLICATION POLYPLOID

OF REPETITIVE

CELLS

P. VAN OOSTVELDT Laboratory

of Biochemistry.

DNA IN

OF PZSCIM SATZVUM and R. VAN PARIJS

Fuculty of Agricultural B9000 Ghent. Belgium

Sciences, State University.

SUMMARY Earlier reported results of a difference in the amount of DNA between 2C meristematic plumula cells and 2C cortex cells of the epicotyls are confirmed by fluorometric Feulgen-DNA determinations. Renaturation experiments of isolated DNA suggest that this difference in DNA is probably correlated with an underreplication of some highly repetitive DNA sequences in cells preparing endomitosis.

The growth of the epicotyl in germinating nucleus, correlated with an underrepPisum sativum L. seeds occurs mainly by lication of some highly repetitive DNA cell elongation. Although DNA is syn- sequences. thesized the cortex cells of the epicotyl MATERIALS AND METHODS seem unable to divide, resulting in the formation of polyploid nuclei [39]. During ger- Material mination in the dark the cortex nuclei reach Seeds of the common garden pea Pisum sativum L. an 8C DNA level with respect to the 2C C.V. rondo (Seed-Trade Labor, Ghent, Belgium) were germinated in the dark at 15°C [41]. Tissues of difDNA content of the meristematic cells in ferent stages were fixed. hydrolysed and stained simultaneously in order to obtain comparable relathe plumula [40, 411. tive amounts of DNA per cell. Fixation was performed Apart from these DNA duplications a dif- in AFA (ethanol, formaldehyde 37%. acetic acid, ference was found in the amount of DNA 75: 20: 5). The tissues were dehydrated through tertiary butanol and embedded in paraffin. Slices of between 2C meristematic cells in the I5 pm thickness were cut from ungerminated emplumula and the 2C cortex cells in the epi- bryos, 20 pm slices were taken from epicotyls after 4 and 8 days of germination. The sections were fixed on cotyl, the latter preparing the process of slides with Mayers fixative (an equal amount of glyendomitosis. In this article we present new cerin and egg white albumin). Different stages of germination are shown in fig. I. evidence for the existence of a quantitative For biochemical experiments the part of the stem bedifference in DNA per cell as determined by tween the cotyledons and the first leave (shaded area in fig. I) is used as epicotyl while the other part of the quantitative histophotometric fluorescence stem is used as plumula. measurements. Renaturation experiments with DNA isolated from octaploid tissue Quantitative fluorescence micro(epicotyls after 3 to 4 days germination in spectrophotometry After hydrolysis the slides were stained in 0.01% acrithe dark) point to the conclusion that a dif- flavine-Schiff (Serva, Heidelberg, BRD) or 0.01% ference exists in the amount of DNA per fuchsin-Schiff (G. T. Gurr, London, UK) prepared acExptl Cell Res 5%(1976)

DNA in polyploid

cells

211

Fig. 1. Schematic drawing and microphotograph of the germinating seed of Pisum sotivum showing different stages used in this study. A, Seed just after soaking in water for II h, one cotyledon is removed; B, seed about 18 h germinating at 15°C; C, about 4 day old seedling germinated in continuous dark; D, microphotograph showing a longitudinal section through the seed just after soaking. PI, plumula; Cor, cotyledon, the epicotyl part is shaded: M, plumula metistem; Cep, cortical cells of the epicotyl part; Cr. cortical cells of the root; Vas, vascular tissue of the root. Preparation coloured for DNA with fuchsin Schiff.

cording to Graumann [16]. The staining (1 h at room temperature) was followed by 6 rinses 5 min each. in a freshly prepared SO, bath (200 ml distilled water. IO ml N HCI. IO ml 10% NaHSO,). The slides were dehydrated through ethanol and mounted from xylol in Fluormount (G. T. Gurr, London, UK) n y= I.5 I. The slides were stored in the dark to prevent any fluorescence fading. 14-761819

The histojluorometric

technique

The apparatus used consisted principally of the MPV I-fluorometer (Leitz-Wetzlar. Germany) in an optical arrangement allowing successive and simultaneous illumination with transmitted and incident light [5]. Epi-illumination through a Ploem-Opak illuminator was carried out with monochromatic light isolated Expr/ Cell Res 98 (1976)

2 12

Oostveldt und Parijs

Table I. Summav Isolation

of purity purameters for DNA isolated according E 280“Ill

method

Marmur: ethanol precipitate collected by centrifugation After HAP at 70°C Marmur ethanol precipitate on a glass rod HS-HAP

DNA measurements

Preparations were screened and focused in transmitted light by means of a phase contrast system associated with an intensity adjustable Tungsten lamp. In order to reduce any possible photofading an orange OG2/2 mm filter was used between the field diaphragm and the phase contrast condenser. Preparations stained by fuchsin-Schiff were excited at a wavelength of 540 nm (bandwidth 18 nm). The dichromatic mirror no. 4 of the Ploem-Opak was used in combination with a K610 or an RG2 (4 mm) barrier filter. For acriflavine-Schiff preparations an excitation wavelength of 420 nm (bandwidth 3 nm) was used in combination with the dichromatic mirror no. 3 and a barrier filter K590. The values of the relative amount of DNA per nucleus were classified into frequency histograms with log 2/5 or log 2/10 as class intervals in order to obtain symmetrical normal distributions.

Recording

of absorption

spectra

Spectral absorption curves were obtained with the same MPV I-instrument, mounted as for transmission measurements. The slit width was reduced to 10 U, giving a bandwidth varying between 3 to 10 nm.

In situ denaturation of chromatin

profiles

Denaturation of chromatin in situ was followed by using the fluorescent staining technique with acridine orange [32]. Sections of IO p were obtained with a cryostat microtome. The slides were fixed in absolute ethanol :acetone (I : I. v : v) for 30 min at room temExptl Cell Res 98 (I976 )

methods

E 23”,,“I

E IHI, nm

f’r RNA

0.425 0.485 0.441

0.533 0.530 0.498

20-30

0.365 0.389

0.512 0.534

3

spooled

from a Xenon lamp (XBO 150 W, Osram, Berlin). The use of monochromatic light strongly reduces background fluorescence and by changing the slit width of the monochromator the light intensity can be changed continuously so that photofading is easily controlled. Excitation light was standardized at 390 nm with a uranyl glass (Zeiss type F53, Germany). Small fluctuations in light intensity were corrected by changing the diameter of the illuminating field diaphragm. Homogeneity of the illumination beam was tested with the uranyl glass standard according to Bohm [4].

Quantitative

to dijferent

4 Not detectable

perature. After fixation the slides were transferred to absolute ethanol at 0°C until use (within 2 days). The melting of chromatin was followed after rehydration of the slides through an ethanol series. The chromatin was partially melted by heating different slides in a mixture of I xSSC and 4% formaldehyde (SSC=O.lS M NaCI+O.OIS M sodium citrate, pH 7) for 20 min, at different temperatures. After heating, the slides were treated with an RNase solution (I mg/ ml in I xSSC for I h at 37°C) and again dehydrated through an ethanol series. The slides were. keot in water-free pyridine for 5 min, acetylated in pyridine acetic acid anhvdride (3 : 2. v : v) for 15 min and stained in a 10m4 M a&dine orange solution at 20°C. following the method of Rigler [32]. Using an excitation wavelength of 365 nm, in combination with the dichromatic mirror nr 1 and a barrier filter K490, the ratios of the fluorescence intensities at 587 and 528 nm were determined with the described histofluorometer. The wavelengths of 587 and 528 nm were selected by two interference filters (Schott, Mainz, BRD), which were moving before the photomultiplier window.

DNA isolation The tissue was ground in a mortar with dry ice. DNA was extracted according to the method of Marmur [26] including several RNase and pronase digestions. Between the different purification stens DNA was precipitated with 2 vol.of ethanol. The precipitated DNA was spooled on a glass rod according to Marmur [26] or collected by centrifugation (3 500 g for 5 min). The isopropanol precipitation was omitted. As a control DNA was also isolated by high salt hydroxyapatite column chromatography (HS-HAP). The tissue homogenate was first washed with 70% ethanol followed by acetone washing at 0°C. The pellet was resuspended. usine a Potter-Elvehiem Teflon glass homogenisator, in About 50 ml of asolution of 8 M ureum, I M NaCIO, in 0.24 M ohosnhate buffer pH 6.8. Insoluble particles as starch and cell wall material were removed by low speed centrifugation (IO min. 5000 g). The supernatant was then shaken with about 10 ml of a hydroxyapatite (HAP) suspension (Bio-gel H.T., Biorad, CA) equilibrated with the same buffer. After several washes according to Thomas et al. 1351 the DNA was eluted with 0.4 M rnhosphate buffer (PB).

DNA in polyploid cells

213

lysodeicticus (density 1.73I) was included as reference. The gradient was run at 44770 rpm at 25°C for 48 h.

Micrococcus

Electron microscopy Isolated DNA was spread for electron microscopy according to the method of Kleinschmidt [20]. The length of the DNA molecules was measured on photographs by means of a curvimeter.

Kinetics of DNA reassociation

log DNA-Feulgen/nucleus; ordinate: no. of nuclei measured. Frequency histograms of the fluorometric DNA measurements after staining with fuchsin-Schiff and hydrolysis at 60°C with 1 N HCI. (A) Plumula meristem; (B) epicotyl cortex of ungerminated embryos; (C) epicotyl cortex nuclei after 4 days; (0) after 8 days of germination in continuous dark.

Fig. 2. Abscissa:

RNA contamination of the DNA was checked by comparing the UV absorption at a wavelength of 268.5 nm after hydrolysis of the DNA in 5% TCA (15 mitt at 90°C) [23] and deoxyribose determination [9]. As an estimate of protein contamination the ratios of the extinctions at 260, 280 and 230 were measured. The results of different isolation methods are summarized in table 1. The concentration of the DNA was determined by measuring the absorption of the sheared DNA at 260 nm after purification on HAP. The CJ value was calculated as OD/2xtime in hours [21].

Thermal denaturation of DNA Melting of DNA in solution was followed at 260 nm with a Gilford 2400 spectrophotometer. The temperature of the cuvet chamber was increased, O.l”C/min, by use of a Haake circulating waterbath. The thermal expansion was corrected by using a guanosine solution with the same optical density.

Analytical CsCl gradient centrifuga tion Equilibrium CsCl gradient centrifugation of DNA was carried out as described by Mandel et al. [25]. DNA of

The DNA isolated according to Marmur [26] was suspended in 0.03 M PB and sheared by sonication in an MSE ultrason, 3 times 1 min with 1 min intervals for cooling on ice. The sonicated DNA was deposited on an HAP-column (1 cmx 1.5 cm) at 70°C. Following the procedure of McCallum & Walker [24] the column was eluted successively with 0.12 M, 0. I6 M and 0.3 M PB pH 6.8. The 0.3 M fraction, containing the purified, double-stranded DNA, was melted by heating for 10 min in a boiling waterbath. After heating the solution was rapidly mixed with a tenfold volume of ice-cold bidistilled water and again deposited on the HAP column at 70°C. After elution, the 0.12 M PB fraction (singlestranded DNA) was retained for renaturation studies. After renaturation at different C,r values, the percentage reassociation was determined by passing the DNA solution, after dilution to 0.03 M PB, over an HAP column at 70°C [7]. Single-stranded DNA was eluted with 0.12 M PB, while double-stranded DNA was eluted with 0.3 M PB. The percentage of reassociation was calculated from (1.2A/l.2A+E)X 100, wherein B is the amount of DNA eluted with 0.12 M PB (measured by absorption at 260 nm) and A is the total amount of DNA eluted with 0.3 M PB. A hyperchromicity of 20% was assumed for single-stranded DNA. Radioactivity of [L4C]labelled DNA was estimated after adding 3.5 ml distilled water and 5 ml Instagel to 1.5 ml DNA solution. The difference in the efftciency of counting between the 0.12 M and the 0.3 M PB solutions was not higher than I %. DNA reassociation was also followed by measuring the decrease in absorbance at 260 nm. Solutions of melted DNA were incubated at 60°C and renaturation was followed in function of time. Afterwards the melting profile of the renatured DNA was again recorded.

RESULTS Histojluorometric DNA determinations The results of the fluorometric DNA determinations (fig. 2) confirm earlier results obtained with absorption cytophotometry [40, 411. Diploid meristematic plumula cells contain about 15% more Feulgen stain than the diploid epicotyl cortex cells of the unExptl Cell Res 9.9(1976)

214

Oostveldt und Parijs

Figs 3, 4. Abscissa: time (min); odinare: fluorescence intensity of 2C nuclei. Changes of fluorescence intensity of O-O, epicotyl and X- - -x, meristematic nuclei with hydrolysis time, at @g. 3) 60°C with 1 N HCI; fig. 4) 28°C with 4 N HCI.

germinated embryo (seeds soaked in running tap water for 18 h). A similar difference is found between the 8C cortex nuclei of the fully elongated cortex cells (epicotyl grown for 8 days in the dark) and 8C nuclei of partly elongated cortex cells (epicotyl grown for 4 days in the dark, stage C of fig. 1). In order to show that these differences are not caused by acid sensitivity against HCl hydrolysis, complete hydrolysis curves of diploid nuclei of the plumula and the Exptl Cell Res 98 (1976)

epicotyl cortex were determined. Two conditions of hydrolysis were tested, after staining with fuchsin-Schiff as well as with acriflavine-Schiffi hydrolysis was carried out at 60°C with 4 N HCl [3]. The results of these experiments are represented in figs 3 and 4. The difference in the amount of Feulgen stain is observed at all stages during hydrolysis at 60°C with 1 N HCl. When hydrolysis is performed at 28°C with 4 N HCl the difference in the amount of Feulgen stain is less pronounced, although a statistical significant difference is found at the 5 % level (Student’s t-test) near the absorption maximum of the stain. This less pronounced difference in fluorescence intensity after hydrolysis at 28”C, can be explained by the occurrence of an inner filter effect [30]. The inner filter effect is reduced by excitation at wavelengths which show a lower absorption. For this reason a second series of measurements were carried out on 4- and g-day old epicotyl cortex cells, using an excitation wavelength of 390 nm instead of 420 nm (fig. 5). The difference in the amount of Feulgen between partially elongated cortex cells (4-day old epicotyl) and completely elongated cortex cells (g-day old epicotyl) is clearly demonstrated and is in full agreement with the difference found after hydrolysis at 60°C. The fact that exact doublings from 4C to 8C are found suggests that the inner filter effect is now completely abolished. Absorption

spectra

Histophotometric absorption spectra of 10 plumula and 10 epicotyl cortex nuclei were recorded in order to prove that the differences in staining are not caused by small alterations in absorption characteristics. No significant differences in absorption spectra are noted between both types of nuclei in

DNA in polyploid

215

cells

I

25 -

LC

A

BC

390

I.20 IO

20-

5

c 25 -

n 10

20.

5

%T

ilc

ec

In

Fig. 5. Abscissa: log DNA-Feulgen/nucleus; ordinate: no. of nuclei measured. Frequency histograms of the fluorometric DNA measurements of partially elongated epicotyl cortex

cells (A, B), and fully elongated epicotyl cortex cells (C, D), excited at a wavelength of 420 nm (A, Cl or 390 nm (B. D).

the region where fluorescence excitation was carried out (540 nm for fuchsin-Schiff, 420 nm acriflavine-Schiff staining; fig. 6).

The results of the histochemical study point to the conclusion that a difference exists between the amount of DNA in 2C meristematic plumula cells at one side and the amount of DNA in the 2C epicotyl cortex cells, which prepare an endomitotic cell cycle, at the other side. If this difference in the amount of DNA is really existing, then some qualitative differences in the DNA sequences can be expected. In all probability these differences are more pronounced at 4 days germination in the dark, because at this stage nearly all cortex cells

Chromatin

denaturation

in situ

Piesco & Alvarez [29] reported differences in the amount of Feulgen dye content after kinetin treatment of root cortex cells. These results were ascribed to differences in acid sensitivity of the chromatin, in agreement with a change in the thermal stability of the chromatin as checked by acridine orange staining. In our experiments on chromatin denaturation in situ significant differences were found between epicotyl cortex nuclei and plumula cells (fig. 7). The chromatin of the plumula seems to be less stable against thermal denaturation than chromatin of the epicotyl cortex nuclei. However, this difference does no longer exist when the tissue is fixed in AFA. This fixation procedure seems to denature the chromatin, possibly by a combined action of acid and formaldehyde. Thus differences in the amount of Feulgen stain can not be explained on the base of an altered chromatin structure.

ol 8C.

.

0’. d .

6

6’ ,:

0 .

Fig. .6. Abscissa: wavelength (nm X IO-‘); ordinate: absorbance as % of A,,,. Absorption spectra of acriflavine-Schiff (lef) and fuchsin-Schiff (right) coloured nuclei of (0) epicotyl cortex; or (+) plumula meristem.

216

Oostveldt and Parijs

UV absorption near the bottom of the gradient is caused by optical impurities in the CsCl. DNA melting profiles

The DNA isolated from plumula and epicotyl after 3 or 4 days of germination has a melting point of 86°C in 1XSSC. Some difference in the melting point of both DNAs is found if the DNA is isolated according to Marmur [26] and collected by centrifugation after precipitation with 2 vol of ethanol. However, this difference is no longer present if the DNAs are collected by spooling around a glass rod, after precipitation with ethanol, or if the DNAs are isolated by high salt hydroxyapatite column chromatography. The recuperation of DNA by centrifugation seems to enhance the RNA contamination. The results of the melting experiments are summarized in table 2.

L 04t

60

70

80

90

7. Abscissa: temp. (‘C); ordinate: F,,,/8’juu. In situ denaturation profile of (n) ethanol-acetone; (b) FAA-fixed nuclei. The ratio 530/.5!90is uncorrected for photomultiplier sensitivity. Vertical bars indicate the confidence intervals at the 5% level; X- - -X. plumula meristem; O-0, epicotyl cortex.

Fig.

Fast reassociating

Two types of reassociation experiments were carried out. In a first experiment the reassociation of DNA, isolated from plumula and epicotyl tissue (3- to 4-day old

of the epicotyl are octaploid, but are still “missing” some DNA. Because the “missing DNA” is synthesized afterwards in the completely elongated cortex cells it seems obvious that repetitive DNA sequences can be involved. Analytical gradient

CsCl equilibrium

Res 98 (1976 )

lb95

density

Our CsCI gradients reveal one single, symmetrical peak with a density of 1.695 g/ cme3. No satellite peaks are found and no differences can be seen between DNA isolated from plumula and epicotyl tissues. A densitometric scanning of a gradient of plumula DNA is shown in fig. 8. The high ExprlCell

DNA

8. Densitometric librium centrifugation

Fig.

Microcrocus

tracing of a neutral CsCl equiof plumula DNA. References /ysodeikticus DNA (Q= I .73 I g/cm@).

DNA in polyploid

cells

217

Table 2. Summary of the melting experiments of plumula (Pl) and epicotyl (Ep) DNA T,+J, meltingpoint,Usenu, are,respectively,left hand and right hand standard deviation on TM. AT=u,+a,

Pl4D Ep 4D PI 4D Ep 4D PI 4D Ep 4D PI 4D Ep 4D PI 4D Ep 4D Rep HAP Cot 1 Rep UV Cot 1

Buffer

TM

u,

0,

AT

1xssc 1xssc 1xssc 1xssc I xssc IXSSC 0.12 PB 0.12 PB 0.12 PB 0.12 PB 1xssc 1xssc 1xssc

86.0 84.0 86.0 86.0 86.0 86.0 87.5 87.5 84.6 84.6 79.5 78.6 78.2

3.2 3.4 2.8 4.6 2.2 2.8 3.1 3.1 4.6 5.8 10.0 14.8 8.2

3.0 3.0 2.6 4.0 2.6 2.8 3.5 3.5 3.6 4.1 6.0 6.6 8.2

6.2 6.4 5.4 8.6 4.8 5.6 6.6 6.6 8.2 9.9 16.0 21.4 16.4

Hyperchromicity (%) 29 25 29.9 27.3 31 28 32.1 28.4 30.6 31.8 20 19

Remarks DNA precipitated in ethanol by centrifugation DNA precipitated in ethanol and spooled DNA isolated by high salt HAP Native DNA Sheared DNA DNA renatured in 0.12 M PB DNA renatured in 1xssc

reassociation studies. Because most of the seedlings) was followed by hydroxyapatite chromatography (table 3) and by the DNA is originating from plumula cells the spectrophotometric technique (260 nm) (fig. reassociation as followed by the UV elution 9). Using both methods a difference is pattern, corresponds to the reassociation of found between DNA isolated from the the plumula DNA, while, on the other hand, plumula and DNA isolated from the epi- the reassociation of the labelled DNA corcotyl. The results show that in the 3-day old responds to the reassociation of the DNA epicotyl some rapidly renaturating DNA originating from the endomitotic cells. Reassociation was determined after three fractions are less redundant. C,t values. Total elution patterns different In the second experiment seeds were soaked in running tap water for 18 h and put in germination dishes on moistened cotton O- D.. 0. ‘.’ 0+. wool for another 18 h. Subsequently about ,. -4 b, D *100O* 50 germs were excised under dim green * Oo safelight and incubated with their roots in a 20+*.*t % * 0 +** 0 solution of [14C]thymidine for 18 h (2 pCi/ f. ml). As revealed by histoautoradiography 30most labelled nuclei are present in the corLOtex of the root and epicotyl base, where *I. . these nuclei go through an endomitotic cell 5ocycle. In this way, it is possible to label specifically the endomitotic cells. After in,02 to-' 100 10’ cubation the germs were mixed with a large Fig. 9. Abscissa: c,,r value; ordinate: % renaturation. UV renaturation pattern of DNA isolated from 0, amount of plumula tissue and from this epicotyl or f, plumule after 3 days of germination in material the DNA was isolated and used for thedark.

218

Oostveldt and Parijs

Table 3. Percentage reassociation of plum&a (PI) and epicotyl (Ep) DNA isolated by high salt hydroxyapatite column chromatography from I-day-old seedlings germinated in continuous dark cot

PI

EP

1 6.6 10 100 103

23 21

II 25

31 37

of the columns are shown in fig. 10. The experiments clearly indicate that some rapidly reassociating DNA sequences in the plumula are not replicated in the endomitotic cells. Characterization DNA

of the reassociated

A larger amount of renatured DNA was Cl1 isolated after C,t 1 by hydroxyapatite chromatography for determination of the properties of the DNA hybrid. oil This DNA shows a T,,, of about 79°C in 1xSSC (table 2). According to Ullmann & McCarthy [37] this T, value corresponds Fig. IO. Abscissa: fraction no.; ordinate: (left) extincto a base pair mismatching of about 9%. tion at 260 nm; (right) cpm. We also tested the ability of the repetitive H+P chromatographic profile of [WC]-labelled DNA to form closed, circular structures by pea DNA (x---X) (polyploid cells) renatured in the presence of an excess of plumula DNA (O-IO) at difreassociation at extremely low concentra- ferent Cot values (from top to bottom): 0.1; 1; 10. tions [17, 311. The isolated reassociated DNA (C,,? 1) was diluted to an end condoubtful circular form while 24.5 % are centration of 5 to 10 pg DNA/ml. The DNA present as aggregates or comcatemeres. The was denatured and reassociation was permean length of the circular DNA molecules formed at 60°C in 0.12 M PB overnight. This was about 676 basepairs, while the linear DNA solution was then spread for electron pieces showed an average length of about microscopic observation. 390 basepairs. On random taken photographs of six preparations 1069 molecules were counted. DISCUSSION 53.1% of the DNA molecules show a linear structure, 15.2 %. have a circular structure The process of endomitosis, resulting in the and about 7.3% of the molecules have a formation of polyploid or polytene nuclei, Exptl Cell Res 98 (1976)

DNA in polyploid

can be considered as a normal differentiation process in higher plants [lo, 361. In this article, we present evidence that some DNA fraction is missing in the diploid epicotyl cells, predestinated to undergo endomitosis, in comparison with diploid meristematic cells where the normal 14 chromosomes can be observed. The epicotyl cortex cells double twice their total amount of DNA during elongation in the dark. When the elongation process is finished the “missing” DNA is synthesized so that afterwards the cortex cells obtain exactly a 4-fold amount of DNA in comparison with the normal meristematic cells. These results were found by quantitative histophotometric DNA determinations, which show a difference in Feulgen stain content, between the 2C meristematic and epicotyl cortex cells, and are most convincingly explained by assuming the existence of a real difference in amount of DNA per nucleus. The same results were obtained by absorption spectrophotometry [40, 411 and quantitative fluorescence spectrophotometry. The difference in Feulgen stain was found in seedlings growing in the dark or in the light, although the DNA concentrations in these nuclei are changing at least by a factor 2 (see [41]). The difference is found during hydrolysis at 60°C with 1 N HCl as well as during hydrolysis at 28°C with 4 N HCl. The finding that the difference is less pronounced after hydrolysis at 28°C can arise from the fact that in this case the extinction values of the nuclei are higher, causing an inner filter effect during fluorescence measurements [30]. A higher yield of Feulgen stain per nucleus after hydrolysis at 28°C was already found by several authors [l 1, 14, 381. The absorption spectra of nuclei of the epicotyl cortex and plumula meristems are identical. No differences are detected in

cells

219

the thermal stability of the chromatin after AFA fixation, able to explain differences in the amount Feulgen stain. The differences observed are also difficult to explain by shifts in the replication time of certain sequence, because no DNA synthesis has occurred after 18 h of soaking in running tap water as revealed by histoautoradiography (unpublished results). For these reasons we assume that the diploid meristematic cells of the plumula contain a higher amount of DNA in comparison with the diploid cortex cells of the epicotyl. The study of the qualitative properties of DNA isolated from octaploid tissue (epicotyls of 3- to 4-day old seedlings), still missing a DNA fraction, and of DNA isolated from the plumula cells points to the conclusion that some repetitive DNA sequences are less redundant in the polyploid tissue. It seems therefore obvious to connect the “missing DNA” as observed with histochemical methods in polyploid cells with “missing” highly repetitive DNA sequences, although we do not have direct evidence for such a connection. Nevertheless, the fact that this “missing DNA” is synthesized later on, after the cells are fully elongated, support the idea that the “missing DNA” consists of reiterated sequences. This highly repetitive DNA shows the same density and the same melting profiles as the main band DNA (fig. 8, table 2). Moreover the DNA of peas seems to be rather homogeneous in GC base composition, as deduced from the narrow band width in CsCl and the narrow melting profile. These statements are in agreement with earlier results [ 181. Complete renaturation curves for pea DNA, already published [34], are in agreement with our results. About 30% of the DNA is renatured after C,t 1. Because the Exprl Cell Res 98 (1976)

220

Oostveldt and Parijs

ribosomal cistrons are about 3 900 times repeated in the pea genome [ 191and are not separable from the main band DNA [33] the possibility exists that the differences found in the reassociation experiments can be partly explained by changes in the redundancy of rRNA cistrons. However, Ingle & Sinclair [19] have found that only 0.17% of the total pea DNA consists of rDNA. In this case an underreplication of ribosomal cistrons alone can not explain the large difference in renaturation rates. Several authors have drawn attention to the fact that bacterial DNA contamination can be an important source of error in the study of plant DNA, especially when radioactive precursors are used. However, the effective concentrations of labelled DNA in our renaturation experiments is so low that if the labelled DNA is of bacterial origin, no reassociation could occur even at a Cot 10. As the reaction is driven by the unlabelled sequences the reservation has to be made that unlabelled bacterial DNA can still interfere with the renaturation studies. As Broekaert & Van Parijs [8] have shown the most important bacterial contaminant in pea seeds has a heavy density and should be seen as a satellite in a CsCl gradient. However, no such satellite can be seen in our DNA preparation of plumula tissue (fig. 8). Melting point determination confirms that DNA isolated at C,r 1 is doublestranded with no more than 9% mismatched basepairs. The fact that the isolated molecules can still form circular structures, proves that in the sheared DNA molecule short repetitive sequences are occurring. Underreplication of repetitive DNA during endomitosis was already reported for different organisms [2, 12, 15, 17, 221. Exptl Cell Res 98 (I 976)

Even in plants, differences in the amount of satellite DNA were reported in different organs [28]. These satellite DNAs are composed of repetitive DNA [ 181,although they are more heterogeneous than animal satellite DNA [I]. Nag1 [27] described the process of endomitosis in plants as a short cut in early prophase of a normal mitotic cell cycle. Several authors already suggested a possible role of repetitive DNA sequences, localized near the centromere, in chromosome structure or folding [6, 13,421. Therefore a switching of mitosis to endomitosis by differential DNA amplification seems to be an acceptable working hypothesis. I would like to thank Professor Dr J. De Lev for running analytical CsCl gradients and Dr R. Hua-fl for giving a sampleof Micrococcus lysodeikticus DNA. Thanks are due to Professor J. Fautrez and Dr F. Roels for use of the cryostat in their laboratory.

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Keceived July 9, 1975 Revised version received August 26, 1975

Exprl Cell Res 98 (1976)