Plants

Plants (1986)167:264-274

9 Springer-Verlag1986

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species C.M. Bowman Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ, UK

Abstract. The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58% _+0.56 (SE) in S. oleracea leaves and 4.97% _+0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r(19)=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication. Key words: Aegilops - Chloroplast DNA - DNA, chloroplast and nuclear - Ploidy - Triticum (chloroplast and nuclear genomes).

Introduction In plants, the biogenesis and continued function of plastids requires the close cooperation of nucleAbbreviations: c t D N A - chloroplast DNA; nDNA = nuclear DNA; RuBPCase=ribulose-l,5-bisphosphate carboxylase; DAPI = 4,6-diamidine-2-phenylindole

ar and plastid genomes (for review, see Parthier 1982). For example, the several components which are assembled into the photosynthetic complexes are encoded by genes found in both chloroplast and nucleus. Some aspects of photosynthesis involve multisubunit enzymes such as ATP synthase, or the CO2-fixing enzyme ribulose-l,5-bisphosphate carboxylase (RuBPCase), in which the genes for the different subunits are again located in the chloroplast or the nuclear genome. Although individual peptide components are exclusively encoded by chloroplast or nuclear genes, each complex or holoenzyme contains components encoded by chloroplast and nuclear genes. Even the machinery for the translation of chloroplast DNA (ctDNA)encoded messages is itself chimaeric: the chloroplast ribosomes contain rRNAs encoded in ctDNA and proteins encoded by both genomes (for review, see Dyer 1984). As ctDNA is multicopy, the numbers of interacting chloroplast and nuclear genes in a single cell may differ several hundred-fold. To overcome this disparity, the synthesis and assembly of the different components of these intricate systems may be coordinated at many levels. For example, in developing wheat leaves, the synthesis of the RuBPCase large-subunit and small-subunit is apparently coordinated by translation of stoichiometric amounts of mRNA (Dean and Leech 1982a). In other cases, such as the Chlamydomonas RuBPCase small-subunit (Schmidt and Mishkind 1983) or the Pisum sativum chlorophyll-a/b-binding protein (Cuming and Bennet 1981) excess protein is synthesised, and this is degraded if it is not incorporated into the appropriate complex. There is a similarity in the replication of chloroplasts and ctDNA which implies that there may also be a coarse control operating at the level of the number of gene copies available for interaction. The implication is that in some cell types, there may be a fixed relationship between the number

C.M. Bowman: Proportionality between chloroplast and nuclear genomes in wheat leaves

265

of chloroplast and nuclear genomes. The important similarity between chloroplast and ctDNA replication is that both are controlled by nuclear genes. This is known from studies of albino mutants of barley and Pelargonium which cannot synthesise ctDNA-encoded proteins. They contain normal numbers of undifferentiated plastids (Hagemann and B6rner 1978) which in turn contain ctDNA which is replicating (Hagemann and B6rner 1978) to normal levels (Steele-Scott et al. 1982). In the closely related genera Triticum (wheat) and Aegilops (goatgrass), the nuclear control of plastid replication leads to an almost linear relationship between nuclear ploidy and plastid number per mesophyll cell (see Jellings and Leech 1984; Ikushima 1983). Does the nuclear control of ctDNA replication lead to a parallel correlation between nuclear ploidy and ctDNA levels in these plants? To answer this question directly, the relative proportions of ctDNA and nuclear DNA (nDNA) have been estimated in mesophyll cells of Tritieum and Aegilops species and related to nuclear ploidy. In the first section of this paper, a simple technique for screening ctDNA proportions is examined. Experiments are described which test the possibility of using the difference in digestibility of ctDNA and nDNA by the methylation-sensitive restriction endonuclease PstI (see Kessler et al. 1985) to measure ctDNA as a proportion of nDNA in leaf extracts from higher plants. In the second section, this selective digestion procedure has been used with quantitive dot-blot hybridisation to estimate ctDNA as a percentage of nDNA in mature mesophyll cells of several diploid, tetraploid and hexaploid species of Triticum and Aegi-

collections maintained at the Plant Breeding Institute, Cambridge. Seeds (100-150) were grown in a peat-based potting compost with a 20-h light period (energy fluence rate 190 W . m -2) and at a temperature of 16 ~ C. When the seedlings were 20-25 cm tall, only the apical 10 cm of each leaf was harvested, to ensure that only expanded tissue was analysed (see Dean and Leech 1982 b). Each batch of freeze-dried leaf powder therefore represented at least 100 plants. Samples of the leaf powder (0.05 g) were frozen in liquid nitrogen in a chilled mortar and then pounded while dry (grinding shears the DNA) to weaken the tissue and break open the cells. A few drops of extraction buffer [2% sarkosyl, 50 mM 2-amino-2-(hydroxymethyt)-l,3-propanediol (Tris), 20 m M ethylenediaminetetraacetic acid (EDTA), 1 mg.m1-1 auto-digested pronase, pH 8.0] were added and this immediately froze. The mixture was gently stirred as the tissue thawed, and extra buffer was added. The extremely viscous homogenate and washings, in a total volume of 5 ml, were transferred to a 15-ml siliconed glass centrifuge tube and incubated for 1 h at 37 ~ C. During incubation the leaf fragments became completely white. After addition of 0.1 vol. of 2 M ammonium acetate, the homogenate was extracted three times with aqueous phenol (pH 8.0) and the nucleic acid was precipitated by the addition of 2.5 vol. of ethanol. The precipitate was washed twice with 70% ethanol, vacuum dried, and allowed to dissolve overnight in 0.5 ml TE buffer (10 mM Tris-HC1, 1 m M EDTA, pH 8.0). The above procedure was chosen to avoid the preferential isolation of either ctDNA or nDNA. Isopycnic banding in caesium chloride was excluded since it is unnecessary for these experiments and separates the ctDNA and n D N A extracted from some plants. The concentration of total D N A in the extracts was estimated by the diphenylamine reaction (Giles and Myers 1965). The intactness of the D N A was monitored by fractionation in 0.85% agarose gels and staining with 4,6-diamidine-2-phenylindole (DAPI) using conditions under which R N A does not stain (see Kapu~ifiski and Yanagi 1979).

lops.

Restriction-endonuclease analysis. Samples of leaf total nucleic acid or c t D N A were digested with PstI as described by the suppliers of the enzyme (Bethesda Research Laboratories, Paisley, UK). Digests were fractionated by electrophoresis on 0.85% agarose horizontal slab gels. Gels for optical density scanning were stained with 2 rag. m l - 1 ethidium bromide solution for 30 rain and destained for 60 rain in distilled water, both with shaking.

Materials and methods Isolation of leaf total nucleic acid. For experiments evaluating the selective digestion technique, freeze-dried powder was prepared from expanded ( > 8 cm) deribbed second leaves of spinach (Spinacia oleracea L. cv. Hybrid 102; Samuel Yates Seed Centre, Chesire, UK) (Scott and Possingham 1980) or from the expanded portion (see Boffey et al. 1979) of hexaploid wheat (T. aestivum L. cv. Mardler) harvested 7 d after sowing, as described elsewhere (Bowman and Dyer 1982). The species and varieties used in experiments to determine the proportion of ctDNA in different ploidy levels of wheat and its relatives represented three ploidy levels and six different cytoplasmic types (see Tsunewaki 1980 and Bowman et al. 1983): diploids, T. boeoticum Boiss. (A-cytoplasm), Ae. speltoides Tausch. (S-cytoplasm), Ae. squarrosa L. (D-cytoplasm), Ae. umbellulata Zhuk. (CU-cytoplasm); tetraploids, Ae. ovata L. (M~ T. dicoccoides (Korn) Schweinf. (B-cytoplasm); hexaploids, T. aestivum cvs. Mardler and Chinese Spring (both B-cytoplasm). Seed of all these species came from

Isolation of chloroplast DNA. Chloroplast D N A was isolated from nonaqueously prepared wheat and spinach chloroplasts (Bowman et al. 1983). Wheat ctDNA was also prepared essentially as described by Kolodner and Tewari (1975).

Optical density scann&g and estimate of peak area. Photographic negatives of ethidium-bromide-stained gels were scanned using a spectrophotometer (SP1800; Pye-Unicam, Cambridge, UK) with pen recorder, or an Ultroscan Laser Densitometer (2202; LKB, Bromma, Sweden) attached to a recording integrator (3390A; Hewlett Packard, Avondale, Pa., USA). Areas under the appropriate peaks on the recorded scans were measured on a digitiser (Apple Computer Inc., Cupertino, Cal., USA). Transfer of DNA to nitrocellulose and hybridisation to 32p-labelled probes. Digests of DNA, fractionated by agarose-gel electrophoresis, were transferred to Schleicher and Schull BA85 nitrocellulose sheets according to Wahl et al. (1979). The depnr-

266

C.M. Bowman: Proportionality between chloroplast and nuclear genomes in wheat leaves

ination step was included to ensure efficient transfer of highmolecular-weight D N A ; this consisted of one 15-min incubation in 0.25 M HC1. Sodium hydroxide alone was used as the neutralising solution. After transfer, the nitrocellulose sheets were washed in 10 x SSC (1 x SSC=0.15 M sodium chloride, 15 mM sodium citrate) and baked in a vacuum oven for 2 h at 80 ~ C. Total-nucleic-acid samples were applied to the nitrocellulose using the 'dot-blot' conditions essentially as described by Kafatos et al. (1979). First, samples (80 lal) were incubated overnight at 37 ~ C in 0.3 M NaOH. These conditions denature the D N A and hydrolyse the R N A to mononucleotides (Brownlee 1972). After addition of 20 lal of 5 M ammonium acetate, the 100-lal samples were loaded on to the nitrocellulose under suction. The sample tubes and applied samples were then rinsed through with a further 300 lal of 1 M ammonium acetate. Without further washing, the 'dot-blots' were baked as described above. The D N A (0.1 to 0.2 lag) to be used for probes was labelled with 3zp by nick-translation (Rigby et al. 1977). The prehybridisation and hybridisation medium was: 0.6 M NaC1, 4 m M EDTA, 100 lag.ml-1 carrier DNA, 2% sodium dodecylsulfate (SDS), 10 x Denhardts solution (Denhardt 1966), 20 mM 1,4piperazinediethanesulfonic acid, pH 6.8. After hybridisation overnight at 65 ~ C, the nitrocellulose sheets were rinsed with 2 x SSC at room temperature and unhybridised material was removed by three 30-min washes in 2 x SSC, 0.1% SDS, at 65 ~ C. The sheets were dried and exposed to X-ray film using tungsten intensifying screens at - 80 ~ C for Southern transfers, or at room temperature for dot-blots. To estimate the relative quantity of probe hybridised to each sample, the region of the nitrocellulose containing the sample was located by autoradiography and excised. The radioactivity in the excised nitrocellulose segments was measured by scintillation counting or C~renkov counting under optimised conditions.

Fig. 1. Demonstration of the conditions for selective and complete PstI digestion of ctDNA in total nucleic acid extracts. Samples containing T. aestivum total nucleic acid (tracks a, b and d-h) or ctDNA (track c) were fractionated by agarose-gel electrophoresis either undigested (tracks a, b) or digested with PstI (tracks c-h). Track c contains 0.5 gg ctDNA, tracks a, b and d contain 2.0 lag total DNA. The undiluted total nucleicacid digest (track d) has been diluted 25-fold in tracks e and f a n d 50-fold in tracks g and h. Staining was with DAPI (track a) or ethidium bromide (tracks b-h). The ctDNA bands (P6+ P7) used for quantitation are identified. Hybridisation of the cloned ctDNA fragment P7 to the D N A in tracks b, c and d is shown in the autoradiograph tracks i, j and k, respectively. Exposure to the X-ray film was adjusted to give an equivalent intensity of signal

Results

Assessment of assay conditions. To assay ctDNA in leaf extracts by digestion with PstI, several conditions must be fulfilled. The isolated D N A must be of high molecular weight, and PstI must selectively and completely cleave the c t D N A in the extract to give distinct bands after fractionation on agarose gels. The n D N A must remain sufficiently intact after digestion to be confined to the tops of gel tracks, so that the ctDNA bands are not obscured. For quantitation of D N A bands, the staining, photography and optical density scanning must all be carried out such that the quantity of D N A in the band is proportional to the recorded peak area. The experimental results illustrated by Figs. 1 and 2 show that these conditions can be met. The intactness of the D N A in the total nucleic acid extracts is demonstrated in Fig. 1 tracks a, b and i, in which undigested D N A has been fractionated. As shown in track a, 4,6-diamidine-2-phenylindole (DAPI) stains only a high-molecular-weight band at the top of the track, this is 95% nDNA. This

demonstrates that the extracts contain an insignificant amount of low-molecular-weight DNA. The intactness of the ctDNA component in the extract is shown by the autoradiograph of track b (track i) in which a nick-translated cloned ctDNA fragment (P7) hybridises only with the high-molecularweight D N A band. Comparison of tracks b-d shows that PstI selectively digests the ctDNA in the extract. The bands characteristic of a PstI wheat ctDNA digest (track c), absent from the undigested sample of wheat total nucleic acid (track b), are distinct in the digested sample (track d). In the autoradiograph (track k) the nick-translated ctDNA fragment P7, hybridises only to the fragment P7 in the total nucleic acid, exactly as it does in the ctDNA digest (autoradiograph track j), and not to any undigested material. This demonstrates that the digestion of the ctDNA in the extract is complete. The units of PstI required for complete digestion of each extract are determined by titrating the enzyme and monitoring the digests by hybrid-

C.M. Bowman: Proportionalitybetween chloroplast and nuclear genomesin wheat leaves

267

II

Dilution factor r cL

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Fig. 2. Quantitationof DNA fractionatedin agarose gels. Samples of undigested total T. aestivum DNA were fractionated by agarose-gel electrophoresis and stained with ethidium bromide. Optimal density scans were made from a photographic negativeof the gel and the area under the O.D. peak was plotted against the quantity of DNA in the appropriate band. The arrows indicate the DNA amount in the bands which have been used to quantitate the nDNA component in dilutions of the most concentrated extract

Fig. 3. Optical density scans of leaf total nucleic-acid digests. Leaf total nucleic-acid samples containing 2.5 gg of T. aestivum total D N A (scan a) and 1.5 gg of S. oleracea total D N A (scan b) were digested with PstI, fractionated by agarose-gel electrophoresis and stained with ethidium bromide. Optical density scans were made from photographic negatives of the gels under quantitative conditions. Peaks corresponding to c t D N A PstI fragments are identified, and those used for c t D N A quantitation are marked by arrows

isation as shown above. For all the extracts assayed in this study, 2 units of PstI was sufficient to digest 1 gg of total D N A in 1 h at 37 ~ C. When these conditions are used to cleave the D N A in wheat total nucleic acid extracts with PstI, a distinct triplet band containing c t D N A fragments: P6 ( x 2 ) + P 7 , separates well from the nDNA. Quantitation of these bands was used to estimate the proportion of ctDNA. The proportion of n D N A in the extracts (e.g. track d) was estimated from a dilution series, e.g. 25-fold (tracks e, f) andor 50-fold (tracks g, h).

It was also established that incubation with PstI does not cause an underestimate of the nDNA. The small amount of cleavage which does occur causes the D N A band to broaden, but on scanning, the area under the peak does not differ significantly when compared with that of undigested DNA.

Optical density scanning. The relative quantity of D N A in appropriate bands was measured from their fluorescence intensity after staining with ethidium bromide, by optical density scanning of photographic negatives (see Pulleybank et al. 1977; Malvy 1984). Figure 2 shows that the linear relationship between the area under the recorded D N A peak and the amount of wheat D N A loaded on the gel extends to bands containing 0.4 gg DNA. It can be seen from the arrows marked against the dilution factor (taken from the most concentrated extract), that all estimates of D N A proportion were made from bands containing D N A at concentration well within the linear range of the curve.

Estimates of ctDNA as a percentage of nDNA in T. aestivum and S. oleracea. For estimation of ctDNA content, therefore, total nucleic acid was extracted from expanded leaves, and the intactness of the D N A was checked by DAPI staining. Aliquots containing sufficient D N A to give visible bands on digestion and electrophoresis (i.e. 4 ~tg DNA) were digested for 1 h at 37 ~ C with 2 units of PstI per gg DNA, (i.e. excess, see Fig. 1) appropriate dilutions were made of the digests and 25-gl volumes of replicate digests and dilutions were fractionated in adjacent tracks of agarose slab gels (e.g. Fig. 1, tracks e-h). Figure 3 shows examples of optical density scans from a track such as that shown in Fig. 1 (track d) for T. aestivum, and of a similar scan of total nucleic acid for S. oleracea. The peaks used to estimate the contribution of ctDNA are indicated by arrows: double band P6+single band P7 for T. aestivum (18.4% of the wheat chloroplast genome, Bowman et al. 1981)

268

C.M. Bowman: Proportionality between chloroplast and nuclear genomes in wheat leaves

Table 1. Estimates of c t D N A as percentage n D N A in leaf extracts of T. aestivum and S. oleracea, by selective digestion. Estimates are means, replicates in brackets, +_SE Extract

Dilution factor

T. aestivum

S. oleracea

1

ctDNA as % n D N A 4.2 5.5 4.9 4.6 3.8 5.1

(2) (2) 6.7 (2) (3) (2) (2) (2)

Mean

2 3

10 25 50 /00 25 25

1

25

14.4 (2) 15.6 (2) 14.58___0.56

2

25

/3.8 (2)

4.97+0.36

and single bands P6 + P7 + P8 for S. oleracea (16.3% of the spinach chloroplast genome, Herrmann et al. 1980). Table 1 summarizes the results for replicate digests of three wheat extracts and two spinach extracts. The mean estimates of ctDNA as a percentage of n D N A in expanded leaves of T. aestivum and S. oleracea were 4.97 _+0.36 (SE) and 14.58 + 0.56 (SE), respectively. Estimates of ctDNA as a percentage of nDNA in Triticum and Aegilops species. Seedlings of T. aestirum cv. Mardler and Chinese Spring, of T. dicoceoides, Ae. ovata, Ae. squarrosa, Ae. speltoides, Ae. umbellulata and T. boeoticum were grown and harvested as described in Materials and methods, so that only developmentally equivalent tissue (i.e., fully expanded cells) was sampled. This was neces-

sary to avoid biasing the results, because it is known that the replication of plastids and plastid D N A are both affected by position in the leaf (Boffey et al. 1979; Dean and Leech 1982b) and by environmental factors (see Possingham and Lawrence 1983). Total nucleic acid was extracted from the leaf samples, and the D N A was monitored for intactness by DAPI staining, and digested with PstI. The contribution of c t D N A was estimated from aliquots containing 4 gg D N A as described in the first section. Dilutions of 25-fold and 50-fold were used to estimate the n D N A proportion. The results shown in Table 2 are means of duplicate estimates. The low value obtained by selective digestion of T. dicoceoides D N A is probably a reflection of the poorer quality of the D N A in several extracts from this species. The ctDNA contribution was also screened by dot-blot hybridisation. Alone this method cannot give absolute values, but if an extract of known ctDNA content is used as a standard, quantitative hybridisation to a ctDNA probe can be used to screen extracts from other tissues or species which contain homologous plastid DNA. In these experiments dilutions of the T. aestivum leaf extract 1, containing 5.2% ctDNA (Table 1) were included as internal standards on all filters. Nucleic-acid spot hybridisation has similarly been used for quantitative screening of lymphoid cell lines for viral D N A (Brandsma and Miller 1980). First it was necessary to establish the linear

Table 2. Estimates of c t D N A as percentage n D N A in leaf extracts from several species of Triticum and Aegilops. Values are means estimated from duplicates by selective digestion (D) or means estimated from the number of replicates in brackets, _+SE (analysis of variance) by dot-blot hybridisation (H) Ploidy

Species and variety

Cytoplasmic n D N A 2C type" value (pg)b

Proportion c t D N A as of mesophyll % total leaf n D N A cells in tissue c

D

c t D N A amount per mesophyll cell (pg)a

H D

H

6x

T. aestivum (Mardler) T. aestivum (Chinese Spring)

B B

34.6 34.6

0.50 0.50

4.9 4.8

4.7_+ 0.6 (4) 3.5 _+0.6 (4)

3.39 3.32

3.25 2.42

4x

T. dicoccoides Ae. ovata

B M~

24.5 /8.5

0.53 0.53

2.8 4.1

4.0+0.4 (9) 4.2_+0.3 (14)

/.43

1.85 1.47

2x

Ae. squarrosa Ae. speltoides Ae. umbellulata T. boeoticum

D S Cu A

10.2 11.6 10.1 -

0.53 0.53 0.53 0.53

4.9 4.8 3.8 4.4

5.1 _+0.6 4.4_+0.5 4.3_+0.5 4.3 _+0.5

0.94 1.05 0.73 -

0.98 0.96 0.82

a b c d

eg. Ogihara and Tsunewaki (/982), Bowman et al. (/983) Bennet and Smith (1976) Jellings and Leech (1982) c t D N A as proportion of total leaf n D N A x n D N A 2C value (pg) Proportion of mesophyll cells in leaf tissue

(5) (8) (7) (10)

C.M. Bowman : Proportionality between chloroplast and nuclear genomes in wheat leaves 8-

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Ng DNA per clot

Fig. 4. Quantitative hybridisation of cloned T. aestivurn ctDNA to dot-blots of T. aestivum leaf total nucleic acid. Replicate samples containing T. aestivurn total nucleic acid were applied to nitrocellulose using the conditions of Kafatos et al. (1979), and hybridised with the nick-translated cloned T. aestivurn ctDNA fragment P7. The quantity of P7 hybridising to each sample was determined by Cerenkov counting

range and sensitivity of the dot-blot method when applied to these plant nucleic-acid extracts. Replicate samples containing 12 ng to 600 ng total D N A per dot were hybridised to nick-translated, cloned wheat c t D N A fragment PT. [In digests of all the extracts, this plasmid hybridised only to the fragment P7 itself (e.g. Fig. 1, track k). Therefore, unless a contaminating species of homologous D N A co-migrates with P7, it is extremely unlikely that the P7-plasmid is hybridising significantly to any homologous nuclear D N A or mitochondrial D N A sequences in the dot-blots.] It was found that in all experiments, up to 0.3 gg total D N A per dot could be applied to the nitrocellulose using the conditions of Kafatos et al. (1979) without affecting the proportionality between the quantity of D N A loaded and the amount of hybridisation (Fig. 4). For routine estimates, therefore, each extract was diluted 50-fold, and duplicate 20-gl, 10-gl (and sometimes 5-gl) aliquots were alkali-treated, applied to the filters, and hybridised with P7. Data was accepted only from those dots giving cpm appropriate to their dilution. After taking into account the sample D N A concentration, the contribution of c t D N A was estimated by reference to the internal standards on each filter. Mean estimates of c t D N A as a percentage of n D N A pooled from three experiments using the dot-blot hybridisation procedure are listed in Table 2 adjacent to the estimates obtained by selective digestion. In general, the two types of estimate

269

agree well, the only exception being the T. dicoccoides values. (Because of the poorer quality of D N A in the T. dicoccoides extracts, the higher values, estimated by the hybridisation procedure from two extracts and nine replicates, are the more realistic, since dot-blot hybridisation is much less sensitive to D N A quality. The 2.8% estimate is therefore considered invalid and will not be discussed further.) Analysis of variance demonstrates that between different species, none of the percentage-ctDNA estimates obtained by the hybridisation procedure is significantly different. Replication of the selective-digestion data was insufficient for statistical analysis, but when the replication is equivalent, e.g. in Table 1, the experimental error is comparable to that of the hybridisation procedure. Therefore, it is improbable that any of the mean values obtained by selective digestion is significantly different. In summary, the estimates of ctDNA as a percentage of n D N A in the species of Triticum and Aegilops analysed, fell within the range 3.5% to 5.1% (Table 2). Given the error inherent in this type of experiment, the variation observed between cytoplasms and species is probably not significant. The trend is however clear: that there is no marked change in ctDNA proportion associated with polyploidy in these species. The range of percentagectDNA levels among the diploid species (3.8% to 5.1%) is essentially the same as that found among the polyploid species (3.5% to 4.9%). The species used in this study were chosen for several reasons. Among them is the availability of useful parameters which have been established from detailed studies of their leaf development, for example: the proportion of mesophyll cells in the expanded leaf, the average number of chloroplasts per mesophyll cell, the region of the leaf containing expanded cells (Boffey et al. 1979), the fact that the first leaves are > 9 0 % 2C (Ellis et al. 1983), and the 2C amount of n D N A (for other references see Tables 2 and 3). A knowledge of these parameters makes it possible to use the percentage-ctDNA estimates to calculate other values such as amounts of ctDNA per mesophyll cell, and ctDNA copy number per mesophyll cell and per chloroplast. While such data extrapolation is valuable, it must be remembered that each time an estimate is used in the calculations, the error is compounded. The amounts of ctDNA per mesophyll cell are shown in the final columns of Table 2. In Table 3 are the calculated c t D N A copy numbers per mesophyll cell and per haploid genome. These data re-

270

C.M. Bowman: Proportionality between chloroplast and nuclear genomes in wheat leaves

Table 3. Calculation of ctDNA copy numbers for several species of Triticum and Aegilops. Copy numbers are calculated from the ctDNA amount per mesophyll cell values shown in Table 2, on the basis that: I copy of wheat ctDNA (135 kbp, 90.106 daltons) weighs 1.49.10-14 g. The original estimates of ctDNA as percentage n D N A from which these figures derive, were obtained either by selective digestion (D) or by dot-blot hybridisation (H) Ploidy

Chloroplast number per mesophyll cell"

Species and variety

6x

T. aestivum (Mardler) T. aestivum (Chinese Spring)

4x

T. dieoceoides Ae. ovata

98 94

Ae. squarrosa Ae. speltoides Ae. umbellulata

57 62 66

2x

155 130

Average number of ctDNA copies per mesophyll cell

per haploid genome

per chloroplast

D

H

D

H

D

H

22750 22280

21810 16240

3790 3710

3640 2710

146 171

141 125

9600

12420 9870

2400

3105 2470

102

127 105

6310 7050 4900

6580 6440 5500

3160 3530 2450

3290 3220 2750

111 114 74

115 104 83

a Jellings and Leech (1984) and personal communication

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nDNA per mesophyll cell (pg)

5'0

160

150

260

Chloroplasts per mesophyll cell

Fig. 5a, b. Relationships between ctDNA amount and n D N A amount (a) or number of chloroplasts (b), in mesophyll cells of several Tritieum and Aegilops species. When (a) values for ctDNA amount per mesophyll cell are plotted against n D N A amont per mesophyl] cell (Bennett and Smith 1976), regression analysis demonstrates a strong linear relationship (ro9)= -[-0.935, P>0.01). The regression line is shown. When (5) ctDNA amount per mesophyll cell is plotted against the average number of chloroplasts per mesophyll cell (Jellings and Leech 1984 and personal communication) there is not such proportionality. This is evident from comparison with the dotted line, which describes the theoretical 1 : 1 relationship

flect the similarity of the percentage-ctDNA estimates, and demonstrate a linear relationship between the amount of ctDNA (and hence ctDNA copy number) and the amount of nDNA per mesophyll cell. This is illustrated by Fig. 5a. In this figure, values for ctDNA amount per mesophyll cell have been calculated from the selective-digestion percentage-ctDNA estimates as shown in Ta-

ble 2. To give the clearest picture of the relationship, calculations were also made from the separate experimental mean values of percentage-ctDNA obtained by the hybridisation procedure. For simplicity,' these were originally pooled to give the dothybfidisation estimates shown in Table 2. Regression analysis of these data shows that in the mesophyll cells of these species of Triticum

C.M. Bowman:Proportionalitybetweenchloroplastand nucleargenomesin wheatleaves and Aegilops, the amount of ctDNA (and consequently the ctDNA copy number) is proportional to the amount of nDNA with a correlation coefficient of 0.935 (19 Degrees of Freedom). The results of Fig.5 b indicate that in these same species, there is no such proportionality between ctDNA amount and chloroplast number per mesophyll cell. While the increase in ctDNA apparently keeps pace with the addition of nuclear genomes during polyploidisation, the chloroplast number increases on average by only 80% with each nuclear-genome addition (Jellings and Leech 1984 and personal communication). Therefore, as shown in Fig. 5 b, there is an increase in the ctDNA amount relative to the chloroplast number with increasing nuclear ploidy. The resulting increase in the number of copies of ctDNA per chloroplast can be seen from the last two columns of Table 3.

Discussion

Evaluation of the selective digestion procedure. The experiments described in the first section of this paper show that the ability of the methylationsensitive restriction endonuclease PstI to discriminate between nDNA and ctDNA in some higher plants can provide a convenient assay for the proportion of these DNAs in leaf nucleic-acid extracts. The basis of this discrimination is the extreme difference in the degree of methylation of ctDNA and nDNA in this type of plant. Their nDNA is so highly methylated that it is extremely resistant to cleavage by methylation-sensitive restriction endonucleases (Gruenbaum et al. 1981). In contrast, no higher-plant ctDNA has yet been found to contain methylated nucleotides (Smillie and Krotkov 1960) and, therefore, digestion with methylation-sensitive enzymes is complete. Clearly such a procedure may only be used to assay ctDNA levels in plants or tissues in which nDNA methylation is high. This is easy to test by digesting the nDNA with a methylation-sensitive enzyme, and comparing the molecular weight of the digestion products with that of the undigested nDNA. If the proportion of high-molecularweight DNA is unchanged, the DNA is suitable. Rice is an example of a plant with unsuitable DNA. The rice nuclear genome is small (1.2 pg DNA per 2C nucleus; Bennett and Smith 1976) and rice nDNA is largely digested by PstI (personal communication, T.A. Dyer, Plant Breeding Institute, Cambridge). In this paper, the selective digestion procedure has been evaluated using wheat and spinach leaves,

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because their percentage-ctDNA levels have already been estimated using different methods. In both types of plant, the methylation of nDNA was high enough to make them suitable for this assay, and selective digestion with PstI gave estimates of ctDNA as a percentage of nDNA (Table 1) which agree with those already reported for the same type of leaf material. The estimate of 14.58%_+0.56 (SE) for deribbed > 8-cm-long spinach leaves is in agreement with values obtained for the proportion of ctDNA in total DNA of expanded spinach leaves measured by reassociation kinetics [e.g. 21.1% _+0.7 (SE) (Scott and Possingham 1980) and 14.5% +4.3 (SD) (Tymms et al. 1982)]. Reassociation kinetics and selective digestion procedures both take into account all leaf-cell types when estimating ctDNA proportions. In some procedures, estimates are related only to mesophyll cells. In hexaploid wheat Dean and Leech (1982b) have measured the proportion of ctDNA in the total DNA of mesophyll cells by extracting ctDNA from known numbers of chloroplasts and by determining mesophyll cell ploidy, nDNA content and chloroplast number. They obtained a value of 13%. Because mesophyll cells represent only 50% of the population of living cells in T. aestivum leaves (Jellings and Leech 1982) the contribution of ctDNA to the total leaf DNA is 6.5%. This is close the value of 4.97%_+0.36 reported here. Selective digestion is therefore a means by which leaf extracts from suitable plants may be assayed for ctDNA content, provided sufficient is known about the ctDNA restriction fragments. In common with reassociation-kinetic procedures it has the advantage that intact chloroplasts need not be isolated and the DNA extraction need not be quantitative. However, the extent to which the estimate of ctDNA proportion in the extracts reflects the situation in the leaf, depends finally on the extraction procedure itself: there must be no preferential extraction of ctDNA or nDNA. The procedure described in this paper showed no indication of favouring either ctDNA or nDNA. The efficiency of extraction did not influence the proportion of ctDNA in the extracts. The use of gel fractionation to discriminate between ctDNA and nDNA means that the procedure is not too complex to be used for screening. However, this also means that the extracted DNA must be of high molecular weight and this may be difficult to achieve from some tissues. The sensitivity of the procedure is also limited by the detectability of the bands chosen for quantitation. By comparison, a screening method such as dot-blot

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C.M. Bowman:ProportiOnalitybetween chloroplastand nucleargenomesin wheatleaves

hybridisation is less demanding of the D N A and' more sensitive. However, dot-blot hybridisation alone cannot give absolute estimates, and requires these for reference. Both methods were therefore used to screen the percentage-ctDNA levels in the Triticum and Aegilops species compared in the second part of this paper. Estimation of ctDNA amounts and copy numbers in mesophyll cells of Triticum and Aegilops species. In the experiments examining the relationship between the amounts of nDNA and ctDNA in mesophyll cells, the species were chosen to represent six different cytoplasmic types (see Table 2) to test the generality of any relationship that might emerge. Two varieties of T. aestivum, containing the B-type cytoplasm, were used as examples of hexaploidy. The ideal experimental design would have been to represent this cytoplasm at each ploidy level. Of the two tetraploids, T. dicoccoides has a B-type cytoplasm. Unfortunately the B-cytoplasm has not been unambiguously identified in any diploid of the Triticeae. Therefore, Ae. speltoides ,with the closely-related S-type cytoplasm (see Tsunewaki 1980) was included among the diploids. The results reported in this paper provide no evidence of any significant variation in the percentage-ctDNA levels associated with cytoplasmic type or species. Therefore, any of these diploid species represents the diploid state equally well. Neither was there any marked change in percentagectDNA levels associated with polyploidy. In the four diploids, the ctDNA content ranged between 3.8% and 5.1%. This range was maintained in the tetraploids and hexaploids (Table2). Consequently, nuclear ploidy, or more precisely nDNA amount was found to be proportional to ctDNA amount and hence to the number of ctDNA copies in the mesophyll cells of these species (Fig. 5 a). These conclusions are not in agreement with those of other studies relating nuclear ploidy to the copy number or percentage of ctDNA in expanded mesophyll cells of Triticum and Aegilops species. Dean and Leech (1982b) report that compared with a constant level of 22% ctDNA in mesophyll cells of diploid and tetraploid wheats, there is a marked drop to 13% with hexaploidy. In contrast, Ikushima (1983) concludes that when compared with the ctDNA copy number in diploid wheats (derived from nucleoid numbers per chloroplast) there is a threefold increase in tetraploid species and a sixfold increase in the hexaploids. Such discrepancies are probably due to the directness

of the method for estimating ctDNA proportion, to the choice of species and to the replication and analysis of the data. Proportionality has, however, been reported between nuclear ploidy and ctDNA amount in tobacco mesophyll protoplasts (Smith 1984) and between nuclear ploidy and ctDNA copy number in Chlamydomonas (Whiteway and Lee 1977). There are many possible explanations for a direct correlation between numbers of chloroplast and nuclear genomes in wheat. The most straightforward is based on the close evolutionary relationship between the Triticum and Aegilops species analysed and the observation that nuclear genes are responsible for ctDNA accumulation (Hagemann and B6rner 1978; Steele-Scott et al. 1982). In such closely related species as these, it is likely that the expression of those nuclear genes responsible for ctDNA accumulation will be regulated similarly during development. This in itself would be sufficient to allow ctDNA copies to accumulate to similar levels during leaf development in cells of the same type. In the same way, any diploid c t D N A : n D N A relationship could be maintained during polyploidisation if each added nuclear genome contained doses of equivalent genes whose products were equally capable of replicating the ctDNA of the diploid progenitor. A more complex explanation allows that the proportionality between chloroplast and nuclear genomes may reflect a more stringent control of ctDNA replication. This could result if ctDNA replication were coupled to the nDNA replication cycle by the coordinate expression of the nuclear genes controlling nDNA- and ctDNA-replication. This type of explanation is less likely and is not the general rule, since in a wider context there is no correlation between leaf ctDNA levels and nuclear genome size (e.g. Lamppa and Bendich 1979a). Further evidence against such an explanation comes from the observation that it is possible to dissociate the replication of nDNA, ctDNA and chloroplasts by illuminating expanding spinach leaf discs with different light regimes (see Possingham and Lawrence 1983). It has also recently been shown that ctDNA and nDNA synthesis are not coupled in suspension cells of Nicotiana tabacure, by the selective inhibition of nDNA polymerase with aphidicolin (Heinhorst et al. 1985). Thus the more straightforward interpretation of the data presented in this paper is also the more likely: that the developmental pattern of expression of those nuclear genes responsible for ctDNA accumulation is similar in all the diploids analysed, and that the percentage-ctDNA levels are main-

C.M. Bowman: Proportionality between chloroplast and nuclear genomes in wheat leaves

tained during polyploidisation because the added doses of equivalent genes are expressed coordinately with those of the diploid progenitor. This interpretation explains the relationship observed between copy numbers of nuclear and chloroplast genomes in these species, without invoking a stringent control mechanism. It is therefore consistent with the evidence that ctDNA replication is not coupled with that of nDNA (Possingham and Lawrence 1983; Heinhorst et al. 1985) but is under relaxed control (see Birky 1983). The results described in this paper provide circumstantial evidence that during polyploidisation in wheat, the behaviour of the nuclear genes controlling ctDNA replication is like that described for the wheat RuBPCase small-subunit genes (Dean and Leech 1982b). That is, the accumulation of their product is proportional to the nuclear genome dosage. In the case of the RuBPCase holoenzyme, this means that in wheat the relative number of gene copies for the large and small subunits is constant. In this, the conclusions of this paper differ from those of Dean and Leech (1982b). It also seems that during polyploidisation there is a parallel in, but not a tight coordination of, the expression of the nuclear genes responsible for the replication of ctDNA and chloroplasts. The relationship between nuclear ploidy and ctDNA amount is linear, but the increase in chloroplast numbers with each added nuclear genome is only 80%. The ctDNA copy number per chloroplast therefore increases at higher ploidy levels (Table 3). This is again consistent with the evidence that ctDNA and plastid replication are not coupled, but it is not so easily reconciled with the suggestion that the nuclear regulation of ctDNA levels might be important in the apparent nucleotypic control of chloroplast reproduction (Butterfass 1983). It is difficult to comment on the functional importance of the results for ctDNA copy number (Table 3). Copy numbers of ctDNA per nuclear genome (or n D N A amount), per cell, or per chloroplast vary greatly among different plant types, under different environmental conditions and at different developmental stages (see Possingham and Lawrence 1983), and so far, few general rules governing these parameters have emerged. One of these generalities is the high multiplicity of the chloroplast genome in mesophyll cells. The ctDNA copy number per diploid genome in mature wheat mesophyll cells (5000-7000 copies) is usual (compare spinach 4900 copies, Scott and Possingham 1980; pea 9600 copies, Lamppa and Bendich 1979b). Wheat nuclear genomes are also usual in

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having extensive reiteration of ribosomal DNA sequences (e.g. 6000-15000 copies per diploid gehome) whose transcriptional activity is correlated with the extent of their methylation (see Flavell et al. 1983). In the absence of any equivalent reiteration or methylation of ribosomal DNA sequences in each chloroplast genome, it is possible that the maintenance of large numbers of complete chloroplast genomes is an alterative solution to a similar problem. It ensures that enough ribosomes are provided to support optimal translation of ctDNAencoded messages. It may be important to balance the capacity of the developing mesophyll cell to translate ctDNA- and nDNA-encoded messages (see Possingham and Lawrence 1983). The polyploid species analysed in this study are all products of successful polyploidisation between very closely related, but different species (allopolyploidisation). The balance maintained between chloroplast and nuclear genome copies must therefore be assessed in this light. Such a genome balance may be required for the successful development of polyploid plants, because it minimises disturbance to established chloroplast-nuclear gene interactions. References Bennett, M.D., Smith, J.B. (1976) Nuclear DNA amounts in angiosperms. Phil. Trans. R. Soc. Lond. 274, 227-274 Birky, C.W. Jr. (1983) Relaxed cellular controls and organelle heredity. Science 222, 468-475 Boffey, S.A., Ellis, R.J., Sellden, G., Leech, R.M. (1979) Chloroplast division and DNA synthesis in light-grown wheat leaves. Plant Physiol. 64, 502-505 Boffey, S.A., Leech, R.M. Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves. Plant Physiol. 69, 1387-1391 Bowman, C.M., Bonnard, G., Dyer, T.A. (1983) Chloroplast DNA variation between species of Triticum and Aegilops. Location of the variation on the chloroplast genome and its relevance to the inheritance and classification of the cytoplasm. Theor. Appl. Genet, 65, 247-262 Bowman, C.M., Dyer, T.A. (1982) Purification and analysis of DNA from wheat chloroplasts isolated in non-aqueous media. Anal. Biochem. 122, 108-I 18 Bowman, C.M., Koller, B., Delius, H., Dyer, T.A. (1981) A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase. Mol. Gen. Genet. 183, 93-101 Brandsma, J., Miller, G. (1980) Nucleic acid spot hybridisation: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc. Natl. Acad. Sci. USA 77, 6851-6855 Brownlee, G.G. (1972) Determination of sequences in RNA. North-Holland publishing Co., Amsterdam London Butterfass, Th. (1979) Patterns of chloroplast reproduction. Cell biology monographs, vol. 6. Springer, Wien New York Butterfass, Th. (1983) A nucleotypic control of chloroplast reproduction. Protoplasma 118, 71-74

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Cuming, A.C., Bennett, J. (1981) Biosynthesis of the light-harvesting chlorophyll a/b protein. Control of messenger RNA activity in light. Eur. J. Biochem. 118, 71-80 Dean, C., Leech, R.M. (1982a) The coordinated synthesis of the large and small subunits of ribulose bisphosphate carboxylase during early cellular development within a seven day wheat leaf. FEBS Lett. 14, 113-116 Dean, C., Leech, R.M. (1982b) Genome expression during normal leaf development 2. Direct correlation between ribulose bisphosphate carboxylase content and nuclear ploidy in a polyploid series of wheat. Plant Physiol. 70, 1605-1608 Denhardt, D. (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Comm. 23, 641 645 Dyer, T.A. (1984) The chloroplast genome: its nature and role in development. In: Chloroplast biogenesis, pp. 23-69, Baker, N.R., Barber, J. eds. Elsevier Science Publishers BV, Amsterdam Ellis, J.R., Jellings, A.J., Leech, R.M. (1983) Nuclear DNA content and the control of chloroplast replication in wheat leaves. Planta 157, 376-380 Flavell, R.B., O'Dell, M., Thompson, W.F. (1983) Cytosine methylation of ribosomal RNA genes and nucleolus organiser activity in wheat. In: Kew Chromosome Conference II, pp. 11-17, Brandham, P.E., Bennet, M.D., eds. George, Allen & Unwin, London Giles, K.W., Myers, A. (1985) An improved diphemylamine method for the estimation of deoxyribonucleic acid. Nature 206, 93 Gruenbaum, Y., Naveh-Many, T., Cedar, H., Razin, A. (1981) Sequence sepcificity of methylation in higher plant DNA. Nature 292, 860-862 Hagemann, R., B6rner, T. (1978) Plastid ribosome-deficient mutants of higher plants as a tool in studying chloroplast biogenesis. In: Chloroplast development, pp. 705-720, Akoynnoglou, G., Argyroudi-Akoyunoglou, J.H. eds. Elsevier/ North-Holland Biomedical Press, Amsterdam Heinhorst, S., Cannon, G., Weissbach, A. (1985) Plastid and nuclear DNA synthesis are not coupled in suspension cells of Nicotiana tabacum. Plant Mol. Biol. 4, 3 12 Herrmann, R.G., Whitfeld, R.R., Bottomley, W. (1980) Construction of a SalI/PstI restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis. Gene 8, 179 191 Ikushima, T. (1983) Reiteration of chloroplast genome in wheat. In: Proc. 6th Int. Wheat Genet. Syrup., pp. 529-535, Sakamoto, S. ed. Plant Germplasm Institute, Kyoto University, Japan Jellings, A.J., Leech, R.M. (1982) The importance of quantitative anatomy in the interpretation of whole leaf biochemistry in species of Triticum, Hordeum and Arena. New Phytol. 92, 39-48 Jetlings, A.J., Leech, R.M. (1984) Anatomical variation in first leaves of nine Triticum geuotypes, and its relationship to photosynthetic capacity. New Phytol. 96, 371-382 Kafatos, F.C., Jones, C.W., Efstradiatis, A. (1979) Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridisation procedure. Nucleic Acids Res. 7, 1541-1552 Kapugciflski, J., Yanagi, K. (i 979) Selective staining by 4,6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels. Nucleic Acids Res. 6, 3535-3542

Kessler, C., Neumaier, P.S., Wolf, W. (1985) Recognition sequences of restriction endonucleases and methylases - a review. Gene 33, 1-102 Kolodner, R., Tewari, K.K. (1975) The molecular size and conformation of the chloroplast DNA from higher plants. Biochim. Biophys. Acta 402, 37~390 Lamppa, G.K., Bendich, A.J. (1979a) Chloroplast DNA sequence homologies among vascular plants. Plant Physiol. 63, 660-668 Lamppa, G.K., Bendich, A.J. (1979b) Changes in chloroplast DNA levels during development of pea (Pisum sativum). Plant Physiol. 64, 126-130 Malvy, C. (1984) Quantification of circular DNA breakage by fluorescent scanning of ethidium bromide-stained horizontal gels: application to the effect of intercalating agents. Anal. Biochem. 143, 158-162 Ogihara, Y., Tsunewaki, K. (1982) Molecular basis of the genetic diversity of the cytoplasm in Triticum and Aegilops. I. Diversity of the chloroplast genome and its lineage revealed by the restriction pattern of ctDNAs. Jap. J. Genet. 57, 371-396 Parthier, B. (1982) The cooperation of nuclear and plastid gehomes in plastid biogenesis and differentiation. Biochem. Physiol. Pflanzen 177, 283-317 Possingham, J.V., Lawrence, M.E. (1983) Controls to plastid division. Int. Rev. Cytol. 84, 1-56 Pulleybank, D.E., Shure, M., Vinograd, J. (1977) The quantitation of fluorescence by photography. Nucleic Acids Res. 4, 1409 1418 Rigby, P.W.J., Dieckmann, M., Rhodes, C., Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 133, 273-251 Schmidt, G.W., Mishkind, M.L. (1983) Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts. Proc. Natl. Acad. Sci. USA 80, 2632-2636 Scott, N.S., Possingham, J.V. (1980) Chloroplast DNA in expanding spinach leaves. J. Exp. Bot. 31, 1081 1092 Smillie, R.M., Krotkov, G. (1960) The estimation of nucleic acids in some algae and higher plants. Can. J. Bot. 38, 31-49 Smith, M.A. (1984) Quantization of chloroplast DNA using dot blot filter hybridisation with double-stranded probes. Mol. Cell. Biochem. 63, 14%156 Steele-Scott, N., Cain, P., Possingham, J.V. (1982) Plastid DNA levels in albino and green leaves of the ' albostrians' mutant of Hordeum vulgare. Z. Pflanzenphysiol. 108, 187-191 Tsunewaki, K. (1980) Genetic diversity of the cytoplasm in Triticum and Aegilops. Japan Society for the Promotion of Science, Japan Tymms, M.J., Steele-Scott, N., Possingham, J.V. (1982) Chloroplast and nuclear DNA content of cultured spinach leaf discs. J. Exp. Bot. 33, 831-837 Wahl, G.M., Stern, M., Stark, G.R. (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridisation by using dextran sulphate. Proc. Natl. Acad. Sci USA 76, 3683-3687 Whiteway, M.S., Lee, R.W. (1977) Chloroplast DNA content increases with nuclear ploidy in Chlamydomonas. Mol. Gen. Genet. 157, 11-15 Received 23 July; accepted 20 September 1985