PHYSIOLOGIA PLANTARUM 85: 625-631. Copenhagen 1992

Comparisoii of three DNA fliiorocliromes for flow cytometric estimatioe of nuclear DNA conteet in

Jaroslav Dolezel, Sergio Sgorbati and Sergio Lucretti

Dolezel, J,, Sgorbati, S. and Lacretti, S. 1992. Comparison of three DNA fluoro- chromes for flow cytometric estimation of nuclear DNA content in plants. - Physiol. . 85:'625-631.

Flow cytometric estimation of nuclear DNA content was performed in sis plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leuko- cytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base prefer- ence were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the spedes-specific differences in binding of these fluorochromes to DNA. !t was coneluded that nuclear DNA content estima- tions performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalating dyes (e.g. propidium iodide) is recommended for this porpose. On the other hand, comparison of the behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA. Key words - DAPI, DNA base content, , mithramycin. naclear DNA content, plants, propidium iodide.

J. Dolelel (corresponding author), Inst. of Experimental Botany, Dept of Plant Biotechnology, Sokolovska 6, CS-77200 Olomoue, Czechoslovakia: S. Sgorbati, Univ. degli Studi di Milano, Dipto di Biologia, Via Celoria 26, 1-20133 Milano, Italy: S. Lucretti, ENEA, C.R.E. Casaccia, C. P. 2400, 1-00100 Roma, Italy.

nucleic acids and include ethidium bromide (EB) and Introduction propidium iodide (PI); and dyes and drugs that show a Estimation of nuclear DNA content is one of the impor- base preference and that include Hoechst 33258 tant applications of flow cytotnetry. The method is (H33258), 4',6-diatnidino-2-phenylindole (DAPI), mi- based on the isolation of single cells or nuclei in suspen- thramycin (MI), chromomycin A3 (CH), and olivomy- sion and on the staining of nuclei with DNA fluoro- cin (OL). chromes. The emitted from each nucleus is As flow cytometry provides only relative values, com- then quantified using a flow cytometer. Although the parison with a reference standard having a known DNA method was originally developed for the analysis of content is necessary to determine picogram quantities of human and cells, it is now widely used also for DNA. To make such a comparison valid, emitted fluo- plants (Galbraith 1989, Dolezel 1991). rescence must be proportional to nuclear DNA contetit Fluorochromes currently used for flow cytometric es- both in a referetice standard and in a sample. WhUe in timation of DNA content can be broadly classified into the case of intercalators (EB, PI) binding to DNA is not two groups: stains that intercalate with double stranded affected by base composition (Le Peeq and Paoletti

Received 3 February, 1992; revised 10 April, 1992

41* Phjsiol, Plant, 85. 1W2 625 1967), DNA-specific dyes (H33258, DAPI) were shown 60 min with 0.5 mg r' DAPI, or 50 mg T' MI, or 50 tng to bind preferentially to - (A-T) rich r' PI 4- 50 mg r' RNAase. The samples were filtered regions of the DNA helix (Muiler and Gautier 1975, through a 15-|im nylon mesh just before measurement. Manzini et al. 1983), and DNA-specific drugs (MI, CH, The whole procedure was performed on ice. OL) were shown to bind preferentially to guanosine- Stained nuclei were analysed with a Leitz MPV-Com- cytosine (G-C) rich regions of DNA (Ward et al. 1965). pact flow cytometer. A 100-W high pressure mercury It is generally agreed that fluorochromes showing lamp was used for excitation. Filterblock A was used for base preference should not be used for estimation of analysis of DAPI fluorescence, filterblock H3 was used nuclear DNA content in cases where base content of a for analysis of Pl fluorescence and filterblock N2 for reference standard and a sample differ (Coleman et al. analysis of MI fluorescence.Histogram s of fluorescence 1981, Muirhead et al. 1985). Such a situation may fre- intensity were registred over 512 channels. All histo- quently occur in plants where overall molar AT or GC grams were evaluated using a Hewlett-Packard HP-86B content is not constant (Shapiro 1976). Nevertheless, microcomputer with FLOWSTAR software (Dolezel while some authors use EB or PI to establish genome 1989). At least 10000 nuclei were analysed in each size in plants (Arumuganathan and Earle 1991, Mi- sample. chaelson et al. 1991a), others prefer to use fluoro- Nuclear DNA content was estimated in relation to an chromes showing base preference such as MI (Galbraith assigned value of 7.0 pg DNA in fresh human leuko- 1990, Kowles et al. 1990) or DAPI (Rayburn et al. 1989, cytes (Tiersch et al. 1989). The leukocytes were used as Rayburn and Auger 1990). One of the probable reasons reference standard for the direct calculation of pico- why base-preference fluorochromes are used is that the gram quantity of DNA in Z, mays and P. sativum. effect of base preference on flow cytometric estimation Because genome sizes of other plant species were con- of nuclear DNA content has so far not been analysed in siderably different from that of leukocytes, Z. mays and detail. P. sativum were used as internal reference for L, escu- In this work, three commonly used DNA fluoro- lentum and V. faba, respectively. Similarly, L. esculen- chromes (PI, DAPI, MI) representing three different tum and F. faba were used as internal reference for types of binding to DNA were compared for flow cyto- estimation of nuclear DNA content in R. sativus and A. metric estitnation of nuclear DNA content in six plant cepa, respectively. This approach was undertaken to species covering a wide range of genome sizes. minimize the risk of error due to nonlinearity and the zero level error (Bagwell et al, 1989, Vindel0v et al. Abbreviations - CH, chromomycin A3; DAPI, 4',6-diami- 1983). dino-2-phenylindole; EB, ethidium bromide; H33258, Hoechst 332.58; MI, mithramycin, OL, olivomycin; PI, propidium io- The ratio of tnodal channel numbers of the G(/G] dide. peaks of the reference standard and sample nuclei (fluo- rescence ratio) was calculated. The ratio was corrected for zero offset error using the DNA ratio between single Materials and methods and double chicken red blood cell nuclei clumps (Givan The plant material was derived from newly expanded et al. 1988). Each sample was analysed 5 times. The leaves of young greenhouse-grown plantlets of radish whole experiment was repeated 4 times. Statistical anal- {Raphanus sativus L.), tomato {Lycopersicon esculen- ysis was performed using Hewlett-Packard software. tum Mill.), maize {Zea mays L.), pea (Pisum sativum L.), broad bean (Vicia faba L.), and onion (Allium cepa L.). Human male leukocytes were used as a primary Results reference standard and chicken red blood cells were The effect of base preference of DAPI and MI on the used for calibration of the flow cytometer. analysis of nuclear DNA content was studied in 6 plant To release plant nuclei, approximately 100 mg of leaf species. They were randomly chosen to cover a wide tissue were chopped with a sharp scalpel in a glass petri range in genome size. Optima! dye concentrations (giv- dish containing 1 ml LBOl lysis buffer (Doiezel et al. ing the smallest coefficients of variation) had been pre- 1989) of the following composition: 15 mMTris, 2 mM viously determined in pilot studies. The coefficients of NajEDTA, 80 mM KCl, 20 mJW NaCI, 0.5 mM sper- variation of GQ/G, peaks ranged from 2.9 to 4.8%, and mine, 15 mM mercaptoethanol, 0.1% Triton X-100, pH were similar for all 3 fluorochromes used in this study. 7.5. Leukocyte nuclei were isolated by resuspending 6 Mean fluorescence ratios are listed in Tab. 1. The X 10' fresh leukocytes in 1 ml ice-cold LBOl buffer. ratios obtained using PI differed from those obtained Because mithramycin (MI) requires magnesium ions for using either DAPI or MI. Genome sizes of selected formation of complexes with DNA (Crissman et al. plant species calculated using fluorescence ratios are 1978), nuclei to be stained with MI were isolated using a listed in Tab. 2. The standard deviations of multiple modified LBOl buffer without NajEDTA and with 50 independent measurements were very small (coeffi- mM magnesium chloride and 30 mM sodium citrate. cients of variation did not exceed 3%) and therefore are The suspensions of released nuclei were passed not listed. In all 6 plant species, genome sizes estimated through a 50-[ifn nylon mesh and stained in the dark for using DAPI differed significantly from those obtained

626 Physiol. Plant,, 85. 1992 Tab. 1. Fluorescence ratios (fluorescenee intensity of a sample/fluorescence intensity of a reference) of G|/G, peaks obtained by flow cytometry using fluorochromes differing in DNA base preference.

Species Fluorescence ratios (mean ± SD, n = 20)

Propidiutn iodide DAPI Mithramycin

R. sativus 0.343±0.005 0.604±0.007 0.488±0.014 L. esculentum 0.569±0.015 0.519±0.{M9 0.590±0.012 Z. mays 0.817±0.006 O.6Ol±0.OO6 1.083±0.02! H. sapiens 1.295 ±0.026 1.432±0.017 I.329±0.025 P. sativum 2.968±0.086 3.068±0.O48 2.946+0.037 V. faba 1.292±0.018 1.631±0.024 1.164±0.009 A. cepa using PI. The differences ranged from —26.4% for Z. ences in binding of base preference fluorochrotnes to mays to -4-44.3% for A. cepa. Similarly, the differences DNA. between genome sizes obtained using PI and MI, re- The values of ratios R calculated for 6 plant species spectively, ranged from —8.2% for yl. cepa to -f96.4% were based on the value of R = 1, arbitrarily chosen for for R. sativus. With the exception of P. sativum and V. human leukocytes (i.e. the 2C DNA content obtained faba, the values were again signiflcantly different frotn using PI and that obtained as the sum of AT -f GC were those obtained using PI. As an example, the rank of PI, considered to be equal in human leukocytes). Thus, for DAPI, and MI fluorescence intensities of Z. mays instance, the value R = 0.974 obtained in Z. mays G,/Gj nuclei relative to leukocyte standard is depicted indicates that among the plant species studied the differ- in Fig. 1. ence in binding of PI and base preference fluoro- To test the possibility that the differences were caused chromes to DNA was smallest in this species. However, by the differences in DNA base composition, AT- and it does not necessarily mean that in Z. mays base prefer- GC-contents were calculated using fluorescence ratios ence dyes were bound less than PI. For instance, choos- obtained with DAPI and MI, respectively. In these ing the value of R = 1.5 for human leukocytes would calculations, a linear base-dependent fluorescence of result in the value of R = 1.461 for Z. mays. To describe base preference dyes was assumed (Leeman and Ruch a difference between a pair of species in binding of PI 1982). As shown in Tab. 3, large differences in overall and in binding of base preference fluorochromes, a ratio AT/GC ratios were observed between individual plant K tnay be used. Its value is independent on the initial species. value chosen for the reference standard. An unexpected finding of this study was that the nuclear DNA content calculated as a sum of AT- and GC-contents (AT + GC) differed from the DNA con- Theoretical considerations tent estimated using PI. When the ratio (R) was calcu- Using flow cytometry, the genome size of any species lated between the DNA content obtained as a sum of can be estimated after simultaneous measuretnent of AT- and GC-contents and the DNA content obtained the fluorescence of stained nuclei of the species and of using PI, it was found to be different in each species the reference standard with known DNA eontent. Let (Tab. 3). This result indicated species-specific differ- FR be the fluorescence ratio and GS, and GS^ genome

Tab. 2. Genome sizes obtained by flow cytometry using propidium iodide (PI), DAPI, and mithramycin (Ml), Human leukocytes (GS = 7.0 pg DNA) served as a primary reference standard. GS = genome size (2C nuclear DNA content in pg); GS,^ |j = GS,c,c,ena ^ fluorescence ratio. The means are based on 20 replicate measurements, the values significantly different (F = 0.01) from GS estimated using PI are marked by an asterisk *. References: (1) Bennett and Smith (1976); (2) Bennett et al. (1982); (3) Van't Hof (1965).

Species PI DAPI MI Published data

GS GS %PI GS %PI GS Reference

R. sativt^s 1.11 1.32* 118.9 2.18* 196.4 0.9 (2) L, esculentum 1.96 2,54* 129.6 3.69* 188.3 2.0 (1) Z, mays 5.72 4,21* 73.6 7.58* 132.5 4.7 (1) F. sativum 9.07 10.02* 110.5 9.30 102.5 9.7 (1) V. fa.ba 26.90 30,75* 114.3 27.41 101.9 26.7 (1) A. cepa 34.76 50.16* 144.3 31.90* 91.8 33.5 (3)

Physiol. Planl, 85. tW2 627 12 Fig. 1. Histograms of nuclear DNA content in maize (M) B and human leukocytes (L) employing fluorescent dyes with different DNA base spe- cificities: A, Propidium io- dide; B, DAPI and C, mith- ramycin. Fluorescence ratio (FR) was ealculated as a ratio of modal channel numbers of the G(/G, peaks of leukocyte 4 • and maize nuclei. Only the FR-O. 817 FR"O. 601 FR-1. 083 first 256 channels of the his- tograms were plotted.

64 128 192 0 64 128 192 0 64 128 192 256 RELATIVE NUCLEAR DNA CONTENT (CHANNEL NUMBER} size of reference standard and of a sample, respectively. equation 3 is not true because GS + (AT + GC). In If the emitted fluorescence is proportional to DNA reality, the fluorescence ratio obtained using a base content the fluorescence ratio is equal to the ratio of the preference fluorochromc is given by genome sizes

GS, B, (AT -1- GC), FR = (4) GS, i; ^AT + GC), and the genome size of a sample can be calculated as where (AT + GC), is the sum of AT and GC content in the reference standard and (AT + GC), is the sum of AT GS, = GS, X FR (2) and GC content in a sample. Let R, = (AT + GC)/GS, In equations 1 and 2. the differences in overall AT/GC and R, = (AT + GC)/GS,. Substituting for (AT -I- ratios are not considered. Therefore, these equations GC)/(AT -f GC), in equation 4 yields cannot be applied to those cases where a DNA fluoro- chrome exhibits base preference and where AT/GC ra- GS, X R, tios in the reference standard and in a sample are differ- FR = — XX (5) ent. In such cases (assuming a linear relationship be- B, GS, X R,, tween the DNA base content and fluorescence intensity) the fluorescence ratio should be given by Let the coefficient K be K=R,,/R,. Substituting for RJR, in equation 5 yields B, GS, FR = — X (3) B, GS, B, GS, (6) where B, is percentage base content (AT or GC) of the reference standard and B, is percentage base content of a sample. From the data in Tab. 2 it is evident that

Tab. 3. Comparison of genome size with the sum of AT and GC contents estimated using flow cytometry. Human leukocytes (AT = 59 5%, Shapiro 1976) were used as a primary reference standard. GS = genome size in pg DNA; FR fluorescence ratio; AT,,,,,^,^ = AT,.,,,.n^ X FR; GC,. „ = GC^,.,,^ x FR; AT (%) = AT/ (AT-hGC) X 100; GC (%) = GC / (AT+GC) x 100; R = (AT -^ GC) / G,S; K — Rjiample ' Rrefereuce.

Species GS AT (pg) AT (%) GC (pg) GC (%) AT-FGC (pg) R K

R. sativus 1.11 0.78 46.99 0.88 53.01 1.66 1.495 0.97 L. esculentum 1.96 1.51 50.17 1.50 49.83 3.01 1.536 1,57 Z. mays 5.72 2.50 44.88 3.07 55.12 5.57 0.974 1.000 0,97 H. sapiens 7.00 4.17 59.50 2.83 40.50 7.00 1.07 P. sativum 9.07 5.97 61,42 3.76 38.58 9.72 1.072 1.093 1.02 V. faba 26.90 18.32 62.31 11.08 37.69 29.40 1.12 A. cepa 34.76 29.88 69.85 12.90 30.15 42.78 1.231

628 Physiol, Plant, 85. ITO2 and thus the genome size of a sample can be calculated ship. Assuming a linear dependence, Kubbies and as follows Friedl (1985) estimated AT-content in human and bo- vine cells. Theit results were in accordance with pub- lished data. Sinailarly, our estimates of AT- and GC- GS, = GS, X FR X :- X - (7) cotitents are also in accordance with data obtained using other techniques (Shapiro 1976, Stack and Comings This conclusion has serious implications. It means that 1979, Leeman and Rach 1982). the genome size of an unknown sample (GSJ cannot be Recently, Brown et al. (1991) suggested a curvilinear correctly estimated using a base preferenee fluoio- relationship between base content and fluorescence in- chrome unless the value of the coefficient K and the tensity of base preference dyes. However, DNA base overall AT/GC ratios in the reference standard and the contents calculated according to their model using our sample are known. fluorescenee ratios differed to various extents, from both our results and published data. For instance the GC-eontent of Allium cepa calculated according to Discussion Brown et al. (1991) assuming 4 GC pairs as a binding The precision and rapidity of flow cytometric estimation site for Ml, was eqtial to 39.64%. This value differs of nuclear DNA content makes the method very attrac- from our result of 30.15%, and also from published tive for estimation of genome size both in animal and values (31.6 and 33.2%) obtained using two different plant species. A range of DNA fluorochromes can be methods (Stack and Comings 1979). Clearly, further used to stain nuelear DNA (Muirhead et al. 1985). research is needed to clarify the relationship. While some of them intercalate quantitatively between Theoretically, the use of fluorochromes showing base base pairs of double stranded nucleic acids, others bind preference should not disturb estimations of nuclear selectively to AT- or GC-rich regions. Insensitivity of DNA content in cases when overall AT/GC ratios in the DNA intercalators (such as EB or PI) to base composi- reference standard and in a sample do not differ tion makes them very suitable for comparative studies (Schnedl et al. 1977, Brown et al. 1991). In contrast, the of DNA content. This was confirmed by the present present study showed that the use of base preference study as 2C nuclear DNA contents estimated using PI in fluorochromes may lead to erroneotis estimations even all 6 plant species were very close to values obtained in these cases. The fact that the ratio R of the sum of AT using Feulgen microspectrophotometry (Tab. 2). + GC content to the 2C DNA content estitnated usinjg A good correlation between the DNA values of 10 an intercalator was different for each species indicated plant species determined by flow cytometry using PI species-specific differences in binding of base prefer- and by Feulgen cytophotometry was also observed by ence fluorochromes to DNA. Therefore nuclear DNA Michaelson et al. (1991b). Nevertheless, their values for content cannot be estimated correctly using base prefer- P. sativum and V. faba differ by about 15% both from ence fluorochromes even in cases where overall AT/GC the data obtained by microspectrophotometry and from ratios in the standard and the sample are equal. our data. Although such discrepancies may be due to The nature of these differences is not clear. Dar- the use of different cultivars of the species, they may be zynkiewicz et al. (1984, 1988) have shown that a large also due to use of improperly calibrated reference stan- proportion of nuclear DNA in native chromatin is un- dards. The ultimate solution of this problem would be stainable, and that the atnotint of unstainable DNA to select a range of standards covering a whole range of varies depending upon chromatin structure. To avoid a genome sizes in plants and to calibrate them against a possible effect of chromatin structure, we have isolated fundamental standard using a reliable chemical assay. cell nuclei from the same type of tissue (newly expanded Our results clearly demonstrated that the use of base leaf of young plantlets) in all 6 plant species. However, preference fluorochromes may lead to serious errors in as gross organization of chromatin in plants was shown the estimation of genome size in plants by flow cytom- to be species-specific (Nagl 1982), we cannot exclude a etry. In most cases, the differences between DNA con- possibility that the differences in binding of base prefer- tents obtained using PI and those obtained using DAPI ence fluorochromes observed in this study were caused or MI were statistically highly significant. We have sug- by the species-specific differences in chromatin struc- gested that these differences were caused by the differ- ture. ences in DNA base composition and by the differences Another factor to be considered is the effect of the in binding of PI and base preference fluorochromes to arrangement of molecule pairs in DNA. For instance DNA. the binding of DAPI in the minor groove of DNA Throughout this study, a linear relationship between requires 3-4 consecutive AT base pairs (Portugal and fluorescence and base content was assumed for base Waring 1988, Wilson et al. 1989). Thus the differences preference fluorochromes. However, there is no gen- in binding of base preferenee fluorochromes to DNA eral agreement on this relationship. The results of Lee- observed in this study may reflect species-specific differ- man and Ruch (1982) obtained with DAPI and chromo- ences in base pair arrangement (for instance in the case mycin A3 suggested a linear or close to linear relation- of DAPI, the differences in the number of AT-rich

Physiol, Plant, 85. W92 629 binding sites). This conclusion is supported by the re- Galbraith, D, W. 1989. Analysis of higher plants by flow sults of Kubbies and Friedl (1985) who observed spe- cytometry and cell sorting. - Int. Rev. Cytol. 116:165-228. - 1990. Flow cytometric analysis of plant genomes. - In cies-specific differences in quenching of H33258 fluo- Methods in Cell Biology (Z. Darzynkiewicz & H. A. Criss- rescence by BrdU-substituted chromatin. man, eds). Vol. 33, pp. 549-562. Academic Press, Inc., San To conclude, the present study clearly shows that flow Diego, CA. ISBN 0-12-564133-8. - , Harkins, K. R., Maddox, 1. M,, Ayres, N. M., Sharma, cytometric estimations of nuclear DNA content in D. P. & Firooxabady, E. 1983. Rapid flow cytometric anal- plants performed with fluorochromes showing base ysis of the cell cycle in intact plant tissues. - Science 220: preference should be interpreted with caution. 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Edited by C, H. Bornman

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