- Email: [email protected]
Treatment of Soybean Cells with Cell Wall Degrading Enzymes Inhibits Nuclear Division but not DNA Synthesis
J. Plant Physiol. Vol. 135. pp. 404 - 408 (1989)
Treatment of Soybean Cells with Cell Wall Degrading Enzymes Inhibits Nuclear Division but not DNA Synthesis)[HONG WANG" 2, ADRIAN 1 2
J. CUTLER I , M. SALEEM I , and LARRY C. FOWKE2
Plant Biotechnology Institute, National Research Council, Saskatoon S7N OW9 and Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada
Received July 1, 1989 . Accepted July 13, 1989
Summary Soybean protoplast cultures were used to study the effects of cell wall degrading enzymes on the cell cycle. Enzyme treatment inhibited nuclear division but not DNA synthesis, leading to alignment (synchrony) of cells at G2 of the cell cycle. The inhibition of nuclear division extended for several hours after the removal of enzymes. Comparison of two enzyme incubation periods (4 h vs. 16 h) revealed that longer enzyme treatment of protoplasts resulted in an earlier and higher rate of cell division for cultured protoplasts. Similar trends were observed for the appearance of preprophase bands of microtubules. On the other hand, protoplasts from the shorter enzyme incubation experienced a longer post-enzyme period, which might have partially reduced the difference in synchrony between the two treatments.
Key words: Soybean [Glycine max (L.) Merr.}, DNA replication, nuclear division, preprophase bands of micro tubules, protoplast preparation (enzyme), synchronization. Abbreviations: BrdU = 5-bromo-2-deoxyuridine; FrdU = 5-fluoro-2-deoxyuridine; MI = mitotic index; PBS = phosphate buffered saline; PPB = preprophase band. Introduction Although enzyme treatment has been widely used for protoplast isolation, its effects on protoplasts in terms of protein and DNA synthesis, and nuclear division are poorly understood. Recently, several reports have shown that enzyme treatments lead to changes in protoplast lipid composition (Browse et aI., 1988), plasma membrane protein phosphorylation (Blowers et al., 1988), the release of phytotoxic factors (Hahne and Lorz, 1988) and the production of active oxygen species (Ishii, 1987). In cereals, cell wall removal has been shown to lead in some cases to oxidative stress (Saleem and Cutler, 1987) and to dramatic changes in the activity of peroxidase (Saleem et aI., 1988) and other enzymes of oxygen metabolism (Cutler et aI., 1989). Recently, cell cycle synchrony has been observed following enzymic digestion of cell walls (Weber et aI., 1986). Plant cells are usually heterogeneous in terms of the cell cycle ex-
* NRCC No. 30492. © 1989 by Gustav Fischer Verlag, Stuttgart
cept at special developmental stages e.g. sporogenesIs In pollen mother cells and early endosperm development. However, synchronized cells are often required for cell cycle studies and may facilitate genetic transformation (e.g. Okada et aI., 1986). Synchronization by protoplast preparation could be particularly interesting since protoplasts have become a widely used tool for plant cell studies (Fowke et aI., 1985). However, it is not clear how this synchronization was achieved. The present investigation examines (1) DNA replication and nuclear division during enzyme treatment and (2) the mechanism of synchronization through protoplast preparation.
Materials and Methods eelliine
The soybean [Glycine max (L.) Merr.] cell line (SB-l) was maintained in 1-B5 medium (Gamborg, 1982) as described by Wang et al. (1989 a).
Inhibition of nuclear division by protoplast isolation
Protoplast isolation and culture Unless otherwise stated the general procedure for protoplast isolation and culture was as follows. Two days after subculture cells were incubated in enzyme solution consisting of 0.6 % cellulase Onozuka R-I0 (Kanematsu-Gosha ltd., CA, USA), 0.2% Driselase (Plenum Scientific Research Inc., Hackensack, NJ, USA), 0.2 % hemicellulase (Rohm and Haas Co. Canada ltd., West Hill, Ont. Canada) and 0.2 % pectinase (Terochem laboratories ltd., Edmonton, Alberta, Canada) in a protoplast culture medium (Kao, 1982) with pH adjusted to 5.8 in darkness (for various periods described below) on a shaker (approx. 50rpm). The resulting protoplasts were filtered through a 48 p.m nylon mesh. After washing twice with the medium, protoplasts were resuspended at a density of 4 x 105 ml- 1 in the same medium and cultured as a thin layer in tissue culture Petri dishes (60 x 15 mm). For immunocytochemical detection of DNA synthesis a labelling reagent (Amersham International, Buckinghamshire, UK) containing BrdU, an analog of thymidine, was added to the enzyme solution or to the protoplast culture medium at a concentration of 0.1%.
405
medium supplemented with 0.43 M sorbitol were used to determine the effect of basal solution.
Effect 0/ enzyme incubation period on nuclear division in cultured protoplasts Cells were incubated in enzyme solution with or without 0.1 % of the labelling reagent for either 4 h or 16 h. Protoplasts were then isolated and cultured as before with or without 10p.gml- 1 aphidicolin in the absence of labelling reagent. Samples were taken at 0, 8, 12, 18,24 and 35h. The overall MI and the frequency of dividing nuclei which had been labelled with BrdU during the enzyme treatment were determined. Since microtubule preprophase bands (PPBs), which characteristically occur before nuclear division in plant cells, were evident in these soybean cells (see also Wang et aI., 1989 a), the percentage of cultured protoplasts with PPBs was determined.
Results and Discussion
Labelling and pulse-labelling of DNA synthesis with BrdU Immunofluorescence microscopy Protoplasts were fixed in 3.7 % formaldehyde at 24°C in a buffer containing 100 mM 1,4-piperazinediethylsulfonic acid (Pipes), 1 mM MgS0 4 and 2mM ethylene glycol-bis-(aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), pH 6.9, for 8h. The fixed protoplasts were attached to coverslips previously coated with poly-l-Iysine (MW 564,000, Sigma Chemical Co., St. louis, MO, USA) and extracted in 1 % Triton X-l00 for 1 h (in fixation buffer). They were then treated with 0.1 % NaBH4 in phosphate buffered saline (PBS) (O.14M NaCI, 2.7mM KCI, 8.0mM Na2HP04 1.5mM KH 2P0 4, pH 7.0) for 6 min and rinsed three times in PBS each three minutes. A 100 p.l aliquot of monoclonal mouse anti-BrdU antibody (ready for use without dilution, Amersham International) was layered on each coverslip and incubated for 50 - 60 min, followed by three rinses in PBS. The coverslip was then treated with 100 p.l fluorescein-conjugated rabbit anti-mouse IgG antibody (Amersham international) diluted by 1: 30 and rinsed three times in PBS. All nuclei were stained with Hoechst 33258 (20 p.g ml- 1 in PBS). Coverslips were rinsed three times in PBS before mounting in PBS containing 2 % n-propyl gallate and 50 % glycerol. For simultaneous staining of microtubules (preprophase bands), a mouse monoclonal anti-chicken tubulin antibody (Sigma) was used at a dilution of 1: 150 along with the primary antibody against BrdU and followed by the secondary antibody described above. Protoplasts were examined using a Zeiss epifluorescence microscope equipped with an FITC filter set ( # 487709) for detection of the antibody staining and a filter set (# 487703) for Hoechst 33258 staining. Micrographs were recorded on XPl-400 film.
DNA synthesis and nuclear division during enzyme treatment To determine the effect of enzyme treatment on DNA synthesis and nuclear division the procedures for protoplast isolation were modified as follows. Cells were suspended in the enzyme solution containing 0.1 % of the labelling reagent. Samples were taken at 1,4 and 12 h, and fixed for immunofluorescence microscopy. Protoplasts were examined for the percentage of BrdU labelled nuclei and mitotic index (MI) (percentage of cells with dividing nuclei). The term «dividing nuclei» refers to nuclei at any stage between prophase, with clearly condensed chromosomes and obscured nucleoli, and telophase, with condensed chromatin. Two additional enzyme solutions, with the same enzymes but with 0.55M sorbitol or 1-B5
BrdU is an analog of thymidine, which can be effectively incorporated into newly synthesized DNA in the presence of FrdU. The BrdU labelled DNA (nucleus) is then visualized by immunofluorescence (Gratzner, 1982). This approach has been successfully used in studies of DNA replication in animal (Gratzner, 1982; Dolbeare et al., 1983; Morstyn et aI., 1983) and plant cells (Wang et aI., 1989 b). It was also observed that nuclear labelling was subject to inhibition by aphidicolin, an inhibitor of DNA polymerase-ex, suggesting that nuclear labelling was due to DNA replication (Wang et aI., 1989 b). DNA synthesis in populations of nuclei thus can be expressed by the percentage of labelled nuclei. The intensity of fluorescence in individual nuclei is proportional to the length of time the nuclei have been in Sphase.
Table 1: Percentages of BrdU labelled nuclei and mitotic index in soybean cells and protoplasts. To determine the enzyme effect on DNA synthesis and nuclear division, cells were incubated for periods indicated in an enzyme solution containing 0.1 % of the labelling reagent. Samples of 0 h contained undigested cells; samples of 1 h consited of cells with partially digested cell walls and samples of 4 and 12 h were spherical protoplasts. They were fixed for immunofluorescence microscopy. For comparison protoplasts, isolated in the absence of BrdU, were cultured in the presence of BrdU and fixed at comparable times to show DNA synthesis during culture.The percentages of BrdU labelled nuclei were determined from 3 random samples, each containing 200 nuclei. The overall mitotic indices were determined from 3 random samples each containing 500 cells. Time in enzyme or medium (h) 0 1 4 12
Freq. of BrdU labelled nuclei in enzyme solut.
Mitotic index in enzyme solution
0 8.4 16.8 33.0
5.5 3.8 0.4 0
(%)
Freq. of BrdU labelled nuclei during culture
(%)
0 9.3 16.5 35.0
406
HONG WANG, ADRIAN J. CUTLER, M. SALEEM, and LARRY C. FowKE
Figs. 1- 4: Labelling of DNA synthesis in soybean cells. Soybean suspension cells were incubated in an enzyme solution supplemented with S-bromo-2-deoxyuridine (BrdU). Protoplasts were then cultured. The first micrograph (a) of each set shows staining of BrdU labelled nuclei with antibody against BrdU and the second micrograph (b) shows nuclei of the same field stained with Hoechst 33258. Note that only nuclei of the cells fluoresced. x 640. Fig. 1: Cells incubated in enzyme solution for 1 h. a) Only one nucleus in this field was labelled with BrdU. b) All the nuclei in the field. Fig. 2: Cells incubated in enzyme solution for 12 h. a) Note the increase in number of BrdU labelled nuclei over Fig. 1 a. b) Nuclei stained with Hoechst 33258. Note that none of them showed mitotic activity. Figs. 3-4: Nuclei labelled during enzyme treatment (12 h) were visualized following 18 h culture in the absence of BrdU. Fig. 3 a :Both nuclei were labelled with BrdU with one at metaphase. b) showing staining with Hoechst 33258. Fig. 4: Five nuclei were in the field (b). The antibody staining reveals: (1) upper single nucleus typically labelled with BrdU; (2) non-labelling of the middle two nuclei (They probably were derived from an earlier division) and (3) lower two daughter nuclei at telophase showing a smaller size with same level of fluorescence.
After 1 h of incubation in the presence of BrdU, a few nuclei were labelled after antibody treatment (Fig. 1 a). After 12 h of incubation, most labelled nuclei showed bright fluorescence (Fig. 2 a). Nuclei labelled during the enzyme treatment were visualized after a period of culture without BrdU (i.e. a chase period) to observe nuclear division (Fig. 3 a). Those nuclei which had divided during the chase were easily recognized since the resulting daughter nuclei were identical in fluorescence and relatively small (Fig. 4 a). In addition, all nuclei (labelled and unlabelled) were visible when stained with Hoechst 33258 (Figs. 1 b-4b).
DNA synthesis and nuclear division during enzyme treatment Cells were converted to protoplasts gradually during the enzyme treatment. Samples of 1 h consisted of partially di-
gested cells. By 4 h most cells had been converted into protoplasts. The incubation of cells in enzyme solution did not affect labelling of nuclei with BrdU (i.e. DNA synthesis), but it dramatically decreased the percentage of cells (with partially digested cell walls) or protoplasts containing dividing nuclei (Table 1). The percentages of nuclei labelled for various periods in the enzyme solution were almost identical to those of protoplasts cultured in medium for the same periods after enzyme incubation. Furthermore, the percentage of labelled nuclei was not affected by different basal solutions (0.55 M sorbitol, the protoplast culture medium or B5 with 0.43 M sorbitol) (data not shown). These observations show that DNA synthesis is not affected either by the enzymes or the composition of the basal solutions. In contrast, nuclear division was greatly affected by the enzyme treatment. By 1 h, the MI had decreased from 5.5 to 3.8 and to 0.4 by 4 h
Inhibition of nuclear division by protoplast isolation Table 2: Effect of enzyme incubation period on mitotic index and percentage of protoplasts with preprophase bands (PPBs) of microtubules during subsequent culture period. Protoplasts were isolated from soybean suspension cells incubated in an enzyme solution containing 0.1 % of the labelling reagent for either 4h (4h-p) or 16h (16h-p). They were cultured and fixed at 0, 8, 12, 18,24 and 35 h. The mitotic index and the percentage of regenerating protoplasts (or cells) with PPBs (% of PPB cells) were independently determined from 3 samples, each containing 500 protoplasts. Time in culture (h) 0 8 12 18 24 35
Mitotic index 4h-p 16h-p 004 0 0.2 4.3 2.7 13.9 10.6 804 6.6 5.3 8.7 7.2
Freq. (%) of PPB cells 4h-p 16h-p 0 0 3.3 21.2 15.1 31.5 19.5 15.1 8.9 13.0 13.5 14.5
(Table 1). All the dividing nuclei at 4 h were at telophase and no earlier stages of division were observed, indicating that initiation of nuclear division was inhibited. Cells incubated with different basal solutions but the same enzyme composition all showed a rapid decrease in the number of dividing nuclei, suggesting again that the decrease was not due to differences in basal solutions. These results explain why the procedure of protoplast isolation would partially synchronize cell division (Weber et ai., 1986). Since nuclear division is inhibited while DNA synthesis is not affected, nuclei would accumulate in G2 stage during enzyme treatment. These nuclei could then enter M phase synchronously during the following culture period.
Effect of different incubation periods on the frequency of nuclear division The above results suggest that the number of nuclei arrested after S-phase would be proportional to the length of time in enzyme solution shorter than cell cycle, which is about 35h for these soybean cells (Chu and Lark, 1976). When protoplasts treated with enzymes for 4 h (4 h protoplasts) or 16 h (16 h protoplasts) were cultured, the predicted four-fold difference in synchrony was not observed although higher percentages of dividing nuclei were observed for the 16 h protoplasts (Table 2). For both 4 hand 16 h protoplasts there was a lag phase between enzyme removal and cell division. After 12 h in culture the MI of 16 h protoplasts reached a maximum of 13.9 while for 4 h protoplasts it reached a peak of 10.6 at 18 h (Table 2) giving a synchrony ratio of 1.3. Similar trends were observed for the frequency of cells with PPBs. Since PPBs appear earlier in the division cycle, the percentage of cells with PPBs was much higher at 8 h than MI, as would be expected. The frequency of PPBs was generally higher than MIs, suggesting a longer time for PPB initiation and development. The nuclei of the protoplasts were inhibited from divisions for the period in the enzyme solution (enzyme period) and a period (post-enzyme period) after the removal of enzymes (Table2). The significance of each period in attaining
407
synchronous divisions differs between protoplasts isolated from different enzyme incubation periods. This may be explained by a simplified calculation in which the level of synchrony is a function of the enzyme period plus the postenzyme period. For protoplasts incubated 4 h in enzyme solution, the enzyme period was 4 h and the post-enzyme period was close to 12h (Table2). Thus the post-enzyme period plays a major role in aligning cells. For protoplasts incubated 16 h in the enzyme solution, the enzyme period was greater (16h), but the post-enzyme period was shorter (close to 8 h). The overall effect of 16 h incubation (16+ 8 == 24) was greater than that of 4 h incubation (4+ 12 == 16) even though the difference between the 4 hand 16 h treatments was reduced by the longer post-enzyme period shown by 4 h protoplasts. As would be expected, the ratio of synchrony (1.3, see above) is close to the ratio of inhibition period (24/16 == 1.5). It is not clear why protoplasts from the shorter incubation period needed a longer period to reenter mitosis. It is possible that after longer incubations, protoplasts were better adapted to the inhibitory conditions of enzyme treatment and had completed other processes required for mitosis. Thus, soon after the removal of enzyme, mitosis was initiated. Results also demonstrate clearly that many more of the dividing nuclei in cultured 16 h protoplasts synthesized DNA during the period of enzyme incubation. The 16 h protoplasts had about 30 % dividing nuclei labelled when examined between 18 - 24 h in culture while 4 h protoplasts had only 3 % of dividing nuclei labelled during enzyme treatment. When 10 p.g ml- 1 aphidicolin was added to the culture medium, MIs were decreased (data not shown); however, the inhibition of DNA replication at this level of aphidicolin was incomplete (Wang et al., 1989 b). The differences between 4 hand 16 h protoplasts were still clearly reflected in the results with a higher peak for 16 h protoplasts and a longer lag phase for 4 h protoplasts. Synchronization of cell division is basically a process of alignment (or enrichment) of cells at a particular stage of the cell cycle. Theoretically it should be possible to align cells at G1, S, G2 or M of the cell cycle by directly inhibiting these phases. In practice, Sand M phases are more vulnerable to inhibition. The alignment of cells at G liS or G2/M boundary can be achieved by a number of methods: (1) change of medium composition, particularly the elimination of a single nutrient (King et al., 1974; Constabel et al., 1977; Gould et al., 1981; Amino et al., 1983; Sharpe et al., 1986); (2) prevention of DNA synthesis with inhibitors such as 5-fluoro-2deoxyuridine (FrdU) and aphidicolin (Kovacs and Van't Hof, 1970; Chu and Lark, 1976; Szabados et al., 1981; Nagata et al., 1982; Eisenbeiser-Engelbrecht, 1985; Nishinari and Syono, 1986); and (3) alternating light and dark regimes (Tamiya, 1966; Lorenzen, 1970; Mitsui et al., 1987). The simple enzymic process of isolating soybean protoplasts inhibits nuclear division but not DNA synthesis; as a result, many protoplasts are arrested at the G2 stage. It is likely that the blockage occurred at the initiation of mitosis (G2/M) since this is a major eucaryotic cell cycle control point. Weber et al. (1986) interpreted their results as indicat-
408
HONG WANG, ADRIAN J. CUTLER, M. SALEEM, and LARRY C. FOWKE
ing that cells were «reset» by enzyme treatment to a common point around the end of mitosis. This conclusion was largely on the basis that «resetting» could be repeated by converting cells to protoplasts during an ongoing cell cycle. However, Weber et al. did not look for DNA synthesis during enzyme digestion and the apparent resetting is consistent with the post-enzyme inhibition period described here. It is not known whether this is a general phenomenon. However, for cells in which such an effect occurs, synchronization could be achieved by manipulating the length of enzyme incubation period. For other studies using plant protoplasts the effect exerted by enzyme treatment may need to be considered in interpreting results. Acknowledgements H. W. is supported by a scholarship from the University of Saskatchewan. The work was partially supported by an operating grant (A 6304) from the Natural Science and Engineering Council of Canada to L. C. F.
References AMINO, S., T. FUJIMURA, and A. KOMAMINE: Synchrony induced by double phosphate starvation in a suspension culture of Catha· ranthus roseus. Physio!. Plant. 59, 393-396 (1983). BLOWERS, D. P., W. F. Boss, and A. J. TREWAVAS: Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion. Plant Physio!. 86, 505-509 (1988). BROWSE, J., C. R. SOMERVILLE, and C. R. SLACK: Changes in lipid composition during protoplast isolation. Plant Science 56, 15-20 (1988). CHU, Y. and K. G. LARK: Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: suitability of the system for genetic studies. Planta 132, 259-268 (1976). CONSTABEL, F., W. G. W. KURZ, K. B. CHATSON, and J. W. KIRKPATRICK: Partial synchrony in soybean cell suspension cultures induced by ethylene. Exp. Cell Res. 105,263-268 (1977). CUTLER, A. J., M. SALEEM, M. A. COFFEY, and M. K. LOEWEN: Role of oxidative stress in cereal protoplast recalcitrance. Plant Cell Tissue and Organ Culture 18, 113-127 (1989). DOLBEARE, F., H. GRATZNER, M. G. PALLAVICINI, and J. W. GRAY: Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Nat!' Acad. Sci. USA 80, 5573-5577 (1983). EISENBEISER-ENGELBRECHT, I.: Reversible blockage of DNA synthesis in two-cell-systems of Riella helicophylla (Bory et Mont.) Mont. by 5-fluoro-2-deoxyuridine - An attempt to synchronize the two cells. J. Plant Physiol. 123,283-288 (1986). FOWKE, L. c., L. R. GRIFFING, MERSEY, B. G. and M. A. TANCHAK: Protoplasts for studies of cell organelles. In: FOWKE, L. C. and F. CONSTABEL (eds.): Plant Protoplasts, pp. 39-52. CRC Press, Boca Raton, FL (1985). GAMBORG, O. L.: Callus and cell culture. In: WETTER, L. R. and F. CONSTABEL (eds.): Plant Tissue Culture Methods, pp. 1-9. National Research Council Canada Prairie Regional Laboratory, Saskatoon (1982). GOULD, A. R., N. P. EVERETT, T. L. WANG, and H. E. STREET: Studies on the control of the cell cycle in cultured plant cells I. Effects of nutrient limitation and nutrient starvation. Protoplasma 106,1-13 (1981).
GRATZNER, H. G.: Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218, 474-475 (1982). HAHNE, G. and H. U:iRZ: Release of phytotoxic factors from plant cell walls during protoplast isolation. J. Plant Physiol. 132, 345-350 (1988). ISHII, S.: Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves. In Vitro Cell & Devel. BioI. 23, 653 - 658 (1987). KAo, K. N.: Plant protoplast fusion and isolation of heterokaryocytes. In: WETTER, L. R. and F. CONSTABEL (eds): Plant Tissue Culture Methods, pp. 49 - 56. Saskatoon, Saskatchewan: Prairie Regional Laboratory, National Research Council Canada (1982). KING, P. J., B. J. Cox, M. W. FOWLER, and H. E. STREET: Metabolic events in synchronized cell cultures of Acer pseudoplatanus L. Plant a 117, 109-122 (1974). KOVACS, C. J. and J. VAN'T HOF: Synchronization of a proliferative population in a cultured plant tissue. J. Cell BioI. 47, 536-539 (1970). LORENZEN, H.: Synchronous cultures. In: HALLDAL, P. (ed.): Photobiology of microorganisms, 187-212, John-Wiley & Sons, London (1970). MITSUI, A., S. CAO, A. TAKAHASHI, and T. ARAI: Growth synchrony and cellular parameters of the unicellular nitrogen-fixing marine cyanobacterium, Synechococcus sp. strain Miami BG 043511 under continuous illumination. Physiol. Plant. 69, 1- 8 (1987). MORSTYN, G., S.-M. Hsu, T. KINSELLA, H. A. GRATZNER, and J. B. MITCHELL: Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody. J. Clin. Invest. 72, 1844-1850 (1983). NAGATA, T., K. OKADA, and 1. TAKEBE: Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes. Plant Cell Rep. 1, 250-252 (1982). NISHINARI, N. and K. SYONO: Induction of cell division synchrony and variation of cytokinin contents through the cell cycle in tabacco cultured cells. Plant Cell Physiol. 27, 147-153 (1986). OKADA, K., 1. TAKEBE, and T. NAGATA: Expression and integration of genes introduced into highly synchronized plant protoplasts. Mol. Gen. Genet. 205, 398-403 (1986). SALEEM, M. and A. J. CUTLER: Stabilizing corn leaf protoplasts with n-propyl gallate. J. Plant Physiol. 128, 479-484 (1987). SALEEM, M., A. J. CUTLER, M. COFFEY, and M. LOEWEN: Role of peroxidase in protoplast development: the activity and molecular forms of peroxidase in mitogenic and non-mitogenic protoplasts. In: PUITE, K. J., J. J. M. DONS, H. J. HUiZING, A. J. KOOL, M. KOORNNEEF, and F. A. KRENS (eds.): Progress in Plant Protoplast Research, pp. 137 -138, Kluwer Academic Publishers, Dordrecht (1988). SHARPE, F. T., Jr., A. C. HALL, and G. W. SCHAEFFER: Phosphatidylcholine and phosphatidylethanolamine content of partially synchronized rice cell suspension cultures. Physiol. Plant. 66, 191-196 (1986). SZABADOS, L., Gy. HADLACZKY, and D. DUDITS: Uptake of isolated plant chromosomes by plant protoplasts. Planta 151, 141-145 (1981). TAMIYA, H.: Synchronous cultures of algae. Ann. Rev. Plant Physiol. 17, 1-26 (1966). WANG, H., A. J. CUTLER, and L. C. FOWKE: High frequencies of preprophase bands in soybean protoplast cultures. J. Cell Sci. 92, 575-580 (1989 a). WANG, H., A. J. CUTLER, M. SALEEM, and L. C. FOWKE: Immunocytochemical detection of DNA synthesis in plant cells. J. Plant Physiol. (in press) (1989 b). WEBER, G., E. DE GROOT, and H.-G. SCHWEIGER: Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.). Planta 168, 273-280 (1986).
Treatment of Soybean Cells with Cell Wall Degrading Enzymes Inhibits Nuclear Division but not DNA Synthesis)[HONG WANG" 2, ADRIAN 1 2
J. CUTLER I , M. SALEEM I , and LARRY C. FOWKE2
Plant Biotechnology Institute, National Research Council, Saskatoon S7N OW9 and Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada
Received July 1, 1989 . Accepted July 13, 1989
Summary Soybean protoplast cultures were used to study the effects of cell wall degrading enzymes on the cell cycle. Enzyme treatment inhibited nuclear division but not DNA synthesis, leading to alignment (synchrony) of cells at G2 of the cell cycle. The inhibition of nuclear division extended for several hours after the removal of enzymes. Comparison of two enzyme incubation periods (4 h vs. 16 h) revealed that longer enzyme treatment of protoplasts resulted in an earlier and higher rate of cell division for cultured protoplasts. Similar trends were observed for the appearance of preprophase bands of microtubules. On the other hand, protoplasts from the shorter enzyme incubation experienced a longer post-enzyme period, which might have partially reduced the difference in synchrony between the two treatments.
Key words: Soybean [Glycine max (L.) Merr.}, DNA replication, nuclear division, preprophase bands of micro tubules, protoplast preparation (enzyme), synchronization. Abbreviations: BrdU = 5-bromo-2-deoxyuridine; FrdU = 5-fluoro-2-deoxyuridine; MI = mitotic index; PBS = phosphate buffered saline; PPB = preprophase band. Introduction Although enzyme treatment has been widely used for protoplast isolation, its effects on protoplasts in terms of protein and DNA synthesis, and nuclear division are poorly understood. Recently, several reports have shown that enzyme treatments lead to changes in protoplast lipid composition (Browse et aI., 1988), plasma membrane protein phosphorylation (Blowers et al., 1988), the release of phytotoxic factors (Hahne and Lorz, 1988) and the production of active oxygen species (Ishii, 1987). In cereals, cell wall removal has been shown to lead in some cases to oxidative stress (Saleem and Cutler, 1987) and to dramatic changes in the activity of peroxidase (Saleem et aI., 1988) and other enzymes of oxygen metabolism (Cutler et aI., 1989). Recently, cell cycle synchrony has been observed following enzymic digestion of cell walls (Weber et aI., 1986). Plant cells are usually heterogeneous in terms of the cell cycle ex-
* NRCC No. 30492. © 1989 by Gustav Fischer Verlag, Stuttgart
cept at special developmental stages e.g. sporogenesIs In pollen mother cells and early endosperm development. However, synchronized cells are often required for cell cycle studies and may facilitate genetic transformation (e.g. Okada et aI., 1986). Synchronization by protoplast preparation could be particularly interesting since protoplasts have become a widely used tool for plant cell studies (Fowke et aI., 1985). However, it is not clear how this synchronization was achieved. The present investigation examines (1) DNA replication and nuclear division during enzyme treatment and (2) the mechanism of synchronization through protoplast preparation.
Materials and Methods eelliine
The soybean [Glycine max (L.) Merr.] cell line (SB-l) was maintained in 1-B5 medium (Gamborg, 1982) as described by Wang et al. (1989 a).
Inhibition of nuclear division by protoplast isolation
Protoplast isolation and culture Unless otherwise stated the general procedure for protoplast isolation and culture was as follows. Two days after subculture cells were incubated in enzyme solution consisting of 0.6 % cellulase Onozuka R-I0 (Kanematsu-Gosha ltd., CA, USA), 0.2% Driselase (Plenum Scientific Research Inc., Hackensack, NJ, USA), 0.2 % hemicellulase (Rohm and Haas Co. Canada ltd., West Hill, Ont. Canada) and 0.2 % pectinase (Terochem laboratories ltd., Edmonton, Alberta, Canada) in a protoplast culture medium (Kao, 1982) with pH adjusted to 5.8 in darkness (for various periods described below) on a shaker (approx. 50rpm). The resulting protoplasts were filtered through a 48 p.m nylon mesh. After washing twice with the medium, protoplasts were resuspended at a density of 4 x 105 ml- 1 in the same medium and cultured as a thin layer in tissue culture Petri dishes (60 x 15 mm). For immunocytochemical detection of DNA synthesis a labelling reagent (Amersham International, Buckinghamshire, UK) containing BrdU, an analog of thymidine, was added to the enzyme solution or to the protoplast culture medium at a concentration of 0.1%.
405
medium supplemented with 0.43 M sorbitol were used to determine the effect of basal solution.
Effect 0/ enzyme incubation period on nuclear division in cultured protoplasts Cells were incubated in enzyme solution with or without 0.1 % of the labelling reagent for either 4 h or 16 h. Protoplasts were then isolated and cultured as before with or without 10p.gml- 1 aphidicolin in the absence of labelling reagent. Samples were taken at 0, 8, 12, 18,24 and 35h. The overall MI and the frequency of dividing nuclei which had been labelled with BrdU during the enzyme treatment were determined. Since microtubule preprophase bands (PPBs), which characteristically occur before nuclear division in plant cells, were evident in these soybean cells (see also Wang et aI., 1989 a), the percentage of cultured protoplasts with PPBs was determined.
Results and Discussion
Labelling and pulse-labelling of DNA synthesis with BrdU Immunofluorescence microscopy Protoplasts were fixed in 3.7 % formaldehyde at 24°C in a buffer containing 100 mM 1,4-piperazinediethylsulfonic acid (Pipes), 1 mM MgS0 4 and 2mM ethylene glycol-bis-(aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), pH 6.9, for 8h. The fixed protoplasts were attached to coverslips previously coated with poly-l-Iysine (MW 564,000, Sigma Chemical Co., St. louis, MO, USA) and extracted in 1 % Triton X-l00 for 1 h (in fixation buffer). They were then treated with 0.1 % NaBH4 in phosphate buffered saline (PBS) (O.14M NaCI, 2.7mM KCI, 8.0mM Na2HP04 1.5mM KH 2P0 4, pH 7.0) for 6 min and rinsed three times in PBS each three minutes. A 100 p.l aliquot of monoclonal mouse anti-BrdU antibody (ready for use without dilution, Amersham International) was layered on each coverslip and incubated for 50 - 60 min, followed by three rinses in PBS. The coverslip was then treated with 100 p.l fluorescein-conjugated rabbit anti-mouse IgG antibody (Amersham international) diluted by 1: 30 and rinsed three times in PBS. All nuclei were stained with Hoechst 33258 (20 p.g ml- 1 in PBS). Coverslips were rinsed three times in PBS before mounting in PBS containing 2 % n-propyl gallate and 50 % glycerol. For simultaneous staining of microtubules (preprophase bands), a mouse monoclonal anti-chicken tubulin antibody (Sigma) was used at a dilution of 1: 150 along with the primary antibody against BrdU and followed by the secondary antibody described above. Protoplasts were examined using a Zeiss epifluorescence microscope equipped with an FITC filter set ( # 487709) for detection of the antibody staining and a filter set (# 487703) for Hoechst 33258 staining. Micrographs were recorded on XPl-400 film.
DNA synthesis and nuclear division during enzyme treatment To determine the effect of enzyme treatment on DNA synthesis and nuclear division the procedures for protoplast isolation were modified as follows. Cells were suspended in the enzyme solution containing 0.1 % of the labelling reagent. Samples were taken at 1,4 and 12 h, and fixed for immunofluorescence microscopy. Protoplasts were examined for the percentage of BrdU labelled nuclei and mitotic index (MI) (percentage of cells with dividing nuclei). The term «dividing nuclei» refers to nuclei at any stage between prophase, with clearly condensed chromosomes and obscured nucleoli, and telophase, with condensed chromatin. Two additional enzyme solutions, with the same enzymes but with 0.55M sorbitol or 1-B5
BrdU is an analog of thymidine, which can be effectively incorporated into newly synthesized DNA in the presence of FrdU. The BrdU labelled DNA (nucleus) is then visualized by immunofluorescence (Gratzner, 1982). This approach has been successfully used in studies of DNA replication in animal (Gratzner, 1982; Dolbeare et al., 1983; Morstyn et aI., 1983) and plant cells (Wang et aI., 1989 b). It was also observed that nuclear labelling was subject to inhibition by aphidicolin, an inhibitor of DNA polymerase-ex, suggesting that nuclear labelling was due to DNA replication (Wang et aI., 1989 b). DNA synthesis in populations of nuclei thus can be expressed by the percentage of labelled nuclei. The intensity of fluorescence in individual nuclei is proportional to the length of time the nuclei have been in Sphase.
Table 1: Percentages of BrdU labelled nuclei and mitotic index in soybean cells and protoplasts. To determine the enzyme effect on DNA synthesis and nuclear division, cells were incubated for periods indicated in an enzyme solution containing 0.1 % of the labelling reagent. Samples of 0 h contained undigested cells; samples of 1 h consited of cells with partially digested cell walls and samples of 4 and 12 h were spherical protoplasts. They were fixed for immunofluorescence microscopy. For comparison protoplasts, isolated in the absence of BrdU, were cultured in the presence of BrdU and fixed at comparable times to show DNA synthesis during culture.The percentages of BrdU labelled nuclei were determined from 3 random samples, each containing 200 nuclei. The overall mitotic indices were determined from 3 random samples each containing 500 cells. Time in enzyme or medium (h) 0 1 4 12
Freq. of BrdU labelled nuclei in enzyme solut.
Mitotic index in enzyme solution
0 8.4 16.8 33.0
5.5 3.8 0.4 0
(%)
Freq. of BrdU labelled nuclei during culture
(%)
0 9.3 16.5 35.0
406
HONG WANG, ADRIAN J. CUTLER, M. SALEEM, and LARRY C. FowKE
Figs. 1- 4: Labelling of DNA synthesis in soybean cells. Soybean suspension cells were incubated in an enzyme solution supplemented with S-bromo-2-deoxyuridine (BrdU). Protoplasts were then cultured. The first micrograph (a) of each set shows staining of BrdU labelled nuclei with antibody against BrdU and the second micrograph (b) shows nuclei of the same field stained with Hoechst 33258. Note that only nuclei of the cells fluoresced. x 640. Fig. 1: Cells incubated in enzyme solution for 1 h. a) Only one nucleus in this field was labelled with BrdU. b) All the nuclei in the field. Fig. 2: Cells incubated in enzyme solution for 12 h. a) Note the increase in number of BrdU labelled nuclei over Fig. 1 a. b) Nuclei stained with Hoechst 33258. Note that none of them showed mitotic activity. Figs. 3-4: Nuclei labelled during enzyme treatment (12 h) were visualized following 18 h culture in the absence of BrdU. Fig. 3 a :Both nuclei were labelled with BrdU with one at metaphase. b) showing staining with Hoechst 33258. Fig. 4: Five nuclei were in the field (b). The antibody staining reveals: (1) upper single nucleus typically labelled with BrdU; (2) non-labelling of the middle two nuclei (They probably were derived from an earlier division) and (3) lower two daughter nuclei at telophase showing a smaller size with same level of fluorescence.
After 1 h of incubation in the presence of BrdU, a few nuclei were labelled after antibody treatment (Fig. 1 a). After 12 h of incubation, most labelled nuclei showed bright fluorescence (Fig. 2 a). Nuclei labelled during the enzyme treatment were visualized after a period of culture without BrdU (i.e. a chase period) to observe nuclear division (Fig. 3 a). Those nuclei which had divided during the chase were easily recognized since the resulting daughter nuclei were identical in fluorescence and relatively small (Fig. 4 a). In addition, all nuclei (labelled and unlabelled) were visible when stained with Hoechst 33258 (Figs. 1 b-4b).
DNA synthesis and nuclear division during enzyme treatment Cells were converted to protoplasts gradually during the enzyme treatment. Samples of 1 h consisted of partially di-
gested cells. By 4 h most cells had been converted into protoplasts. The incubation of cells in enzyme solution did not affect labelling of nuclei with BrdU (i.e. DNA synthesis), but it dramatically decreased the percentage of cells (with partially digested cell walls) or protoplasts containing dividing nuclei (Table 1). The percentages of nuclei labelled for various periods in the enzyme solution were almost identical to those of protoplasts cultured in medium for the same periods after enzyme incubation. Furthermore, the percentage of labelled nuclei was not affected by different basal solutions (0.55 M sorbitol, the protoplast culture medium or B5 with 0.43 M sorbitol) (data not shown). These observations show that DNA synthesis is not affected either by the enzymes or the composition of the basal solutions. In contrast, nuclear division was greatly affected by the enzyme treatment. By 1 h, the MI had decreased from 5.5 to 3.8 and to 0.4 by 4 h
Inhibition of nuclear division by protoplast isolation Table 2: Effect of enzyme incubation period on mitotic index and percentage of protoplasts with preprophase bands (PPBs) of microtubules during subsequent culture period. Protoplasts were isolated from soybean suspension cells incubated in an enzyme solution containing 0.1 % of the labelling reagent for either 4h (4h-p) or 16h (16h-p). They were cultured and fixed at 0, 8, 12, 18,24 and 35 h. The mitotic index and the percentage of regenerating protoplasts (or cells) with PPBs (% of PPB cells) were independently determined from 3 samples, each containing 500 protoplasts. Time in culture (h) 0 8 12 18 24 35
Mitotic index 4h-p 16h-p 004 0 0.2 4.3 2.7 13.9 10.6 804 6.6 5.3 8.7 7.2
Freq. (%) of PPB cells 4h-p 16h-p 0 0 3.3 21.2 15.1 31.5 19.5 15.1 8.9 13.0 13.5 14.5
(Table 1). All the dividing nuclei at 4 h were at telophase and no earlier stages of division were observed, indicating that initiation of nuclear division was inhibited. Cells incubated with different basal solutions but the same enzyme composition all showed a rapid decrease in the number of dividing nuclei, suggesting again that the decrease was not due to differences in basal solutions. These results explain why the procedure of protoplast isolation would partially synchronize cell division (Weber et ai., 1986). Since nuclear division is inhibited while DNA synthesis is not affected, nuclei would accumulate in G2 stage during enzyme treatment. These nuclei could then enter M phase synchronously during the following culture period.
Effect of different incubation periods on the frequency of nuclear division The above results suggest that the number of nuclei arrested after S-phase would be proportional to the length of time in enzyme solution shorter than cell cycle, which is about 35h for these soybean cells (Chu and Lark, 1976). When protoplasts treated with enzymes for 4 h (4 h protoplasts) or 16 h (16 h protoplasts) were cultured, the predicted four-fold difference in synchrony was not observed although higher percentages of dividing nuclei were observed for the 16 h protoplasts (Table 2). For both 4 hand 16 h protoplasts there was a lag phase between enzyme removal and cell division. After 12 h in culture the MI of 16 h protoplasts reached a maximum of 13.9 while for 4 h protoplasts it reached a peak of 10.6 at 18 h (Table 2) giving a synchrony ratio of 1.3. Similar trends were observed for the frequency of cells with PPBs. Since PPBs appear earlier in the division cycle, the percentage of cells with PPBs was much higher at 8 h than MI, as would be expected. The frequency of PPBs was generally higher than MIs, suggesting a longer time for PPB initiation and development. The nuclei of the protoplasts were inhibited from divisions for the period in the enzyme solution (enzyme period) and a period (post-enzyme period) after the removal of enzymes (Table2). The significance of each period in attaining
407
synchronous divisions differs between protoplasts isolated from different enzyme incubation periods. This may be explained by a simplified calculation in which the level of synchrony is a function of the enzyme period plus the postenzyme period. For protoplasts incubated 4 h in enzyme solution, the enzyme period was 4 h and the post-enzyme period was close to 12h (Table2). Thus the post-enzyme period plays a major role in aligning cells. For protoplasts incubated 16 h in the enzyme solution, the enzyme period was greater (16h), but the post-enzyme period was shorter (close to 8 h). The overall effect of 16 h incubation (16+ 8 == 24) was greater than that of 4 h incubation (4+ 12 == 16) even though the difference between the 4 hand 16 h treatments was reduced by the longer post-enzyme period shown by 4 h protoplasts. As would be expected, the ratio of synchrony (1.3, see above) is close to the ratio of inhibition period (24/16 == 1.5). It is not clear why protoplasts from the shorter incubation period needed a longer period to reenter mitosis. It is possible that after longer incubations, protoplasts were better adapted to the inhibitory conditions of enzyme treatment and had completed other processes required for mitosis. Thus, soon after the removal of enzyme, mitosis was initiated. Results also demonstrate clearly that many more of the dividing nuclei in cultured 16 h protoplasts synthesized DNA during the period of enzyme incubation. The 16 h protoplasts had about 30 % dividing nuclei labelled when examined between 18 - 24 h in culture while 4 h protoplasts had only 3 % of dividing nuclei labelled during enzyme treatment. When 10 p.g ml- 1 aphidicolin was added to the culture medium, MIs were decreased (data not shown); however, the inhibition of DNA replication at this level of aphidicolin was incomplete (Wang et al., 1989 b). The differences between 4 hand 16 h protoplasts were still clearly reflected in the results with a higher peak for 16 h protoplasts and a longer lag phase for 4 h protoplasts. Synchronization of cell division is basically a process of alignment (or enrichment) of cells at a particular stage of the cell cycle. Theoretically it should be possible to align cells at G1, S, G2 or M of the cell cycle by directly inhibiting these phases. In practice, Sand M phases are more vulnerable to inhibition. The alignment of cells at G liS or G2/M boundary can be achieved by a number of methods: (1) change of medium composition, particularly the elimination of a single nutrient (King et al., 1974; Constabel et al., 1977; Gould et al., 1981; Amino et al., 1983; Sharpe et al., 1986); (2) prevention of DNA synthesis with inhibitors such as 5-fluoro-2deoxyuridine (FrdU) and aphidicolin (Kovacs and Van't Hof, 1970; Chu and Lark, 1976; Szabados et al., 1981; Nagata et al., 1982; Eisenbeiser-Engelbrecht, 1985; Nishinari and Syono, 1986); and (3) alternating light and dark regimes (Tamiya, 1966; Lorenzen, 1970; Mitsui et al., 1987). The simple enzymic process of isolating soybean protoplasts inhibits nuclear division but not DNA synthesis; as a result, many protoplasts are arrested at the G2 stage. It is likely that the blockage occurred at the initiation of mitosis (G2/M) since this is a major eucaryotic cell cycle control point. Weber et al. (1986) interpreted their results as indicat-
408
HONG WANG, ADRIAN J. CUTLER, M. SALEEM, and LARRY C. FOWKE
ing that cells were «reset» by enzyme treatment to a common point around the end of mitosis. This conclusion was largely on the basis that «resetting» could be repeated by converting cells to protoplasts during an ongoing cell cycle. However, Weber et al. did not look for DNA synthesis during enzyme digestion and the apparent resetting is consistent with the post-enzyme inhibition period described here. It is not known whether this is a general phenomenon. However, for cells in which such an effect occurs, synchronization could be achieved by manipulating the length of enzyme incubation period. For other studies using plant protoplasts the effect exerted by enzyme treatment may need to be considered in interpreting results. Acknowledgements H. W. is supported by a scholarship from the University of Saskatchewan. The work was partially supported by an operating grant (A 6304) from the Natural Science and Engineering Council of Canada to L. C. F.
References AMINO, S., T. FUJIMURA, and A. KOMAMINE: Synchrony induced by double phosphate starvation in a suspension culture of Catha· ranthus roseus. Physio!. Plant. 59, 393-396 (1983). BLOWERS, D. P., W. F. Boss, and A. J. TREWAVAS: Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion. Plant Physio!. 86, 505-509 (1988). BROWSE, J., C. R. SOMERVILLE, and C. R. SLACK: Changes in lipid composition during protoplast isolation. Plant Science 56, 15-20 (1988). CHU, Y. and K. G. LARK: Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: suitability of the system for genetic studies. Planta 132, 259-268 (1976). CONSTABEL, F., W. G. W. KURZ, K. B. CHATSON, and J. W. KIRKPATRICK: Partial synchrony in soybean cell suspension cultures induced by ethylene. Exp. Cell Res. 105,263-268 (1977). CUTLER, A. J., M. SALEEM, M. A. COFFEY, and M. K. LOEWEN: Role of oxidative stress in cereal protoplast recalcitrance. Plant Cell Tissue and Organ Culture 18, 113-127 (1989). DOLBEARE, F., H. GRATZNER, M. G. PALLAVICINI, and J. W. GRAY: Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Nat!' Acad. Sci. USA 80, 5573-5577 (1983). EISENBEISER-ENGELBRECHT, I.: Reversible blockage of DNA synthesis in two-cell-systems of Riella helicophylla (Bory et Mont.) Mont. by 5-fluoro-2-deoxyuridine - An attempt to synchronize the two cells. J. Plant Physiol. 123,283-288 (1986). FOWKE, L. c., L. R. GRIFFING, MERSEY, B. G. and M. A. TANCHAK: Protoplasts for studies of cell organelles. In: FOWKE, L. C. and F. CONSTABEL (eds.): Plant Protoplasts, pp. 39-52. CRC Press, Boca Raton, FL (1985). GAMBORG, O. L.: Callus and cell culture. In: WETTER, L. R. and F. CONSTABEL (eds.): Plant Tissue Culture Methods, pp. 1-9. National Research Council Canada Prairie Regional Laboratory, Saskatoon (1982). GOULD, A. R., N. P. EVERETT, T. L. WANG, and H. E. STREET: Studies on the control of the cell cycle in cultured plant cells I. Effects of nutrient limitation and nutrient starvation. Protoplasma 106,1-13 (1981).
GRATZNER, H. G.: Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218, 474-475 (1982). HAHNE, G. and H. U:iRZ: Release of phytotoxic factors from plant cell walls during protoplast isolation. J. Plant Physiol. 132, 345-350 (1988). ISHII, S.: Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves. In Vitro Cell & Devel. BioI. 23, 653 - 658 (1987). KAo, K. N.: Plant protoplast fusion and isolation of heterokaryocytes. In: WETTER, L. R. and F. CONSTABEL (eds): Plant Tissue Culture Methods, pp. 49 - 56. Saskatoon, Saskatchewan: Prairie Regional Laboratory, National Research Council Canada (1982). KING, P. J., B. J. Cox, M. W. FOWLER, and H. E. STREET: Metabolic events in synchronized cell cultures of Acer pseudoplatanus L. Plant a 117, 109-122 (1974). KOVACS, C. J. and J. VAN'T HOF: Synchronization of a proliferative population in a cultured plant tissue. J. Cell BioI. 47, 536-539 (1970). LORENZEN, H.: Synchronous cultures. In: HALLDAL, P. (ed.): Photobiology of microorganisms, 187-212, John-Wiley & Sons, London (1970). MITSUI, A., S. CAO, A. TAKAHASHI, and T. ARAI: Growth synchrony and cellular parameters of the unicellular nitrogen-fixing marine cyanobacterium, Synechococcus sp. strain Miami BG 043511 under continuous illumination. Physiol. Plant. 69, 1- 8 (1987). MORSTYN, G., S.-M. Hsu, T. KINSELLA, H. A. GRATZNER, and J. B. MITCHELL: Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody. J. Clin. Invest. 72, 1844-1850 (1983). NAGATA, T., K. OKADA, and 1. TAKEBE: Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes. Plant Cell Rep. 1, 250-252 (1982). NISHINARI, N. and K. SYONO: Induction of cell division synchrony and variation of cytokinin contents through the cell cycle in tabacco cultured cells. Plant Cell Physiol. 27, 147-153 (1986). OKADA, K., 1. TAKEBE, and T. NAGATA: Expression and integration of genes introduced into highly synchronized plant protoplasts. Mol. Gen. Genet. 205, 398-403 (1986). SALEEM, M. and A. J. CUTLER: Stabilizing corn leaf protoplasts with n-propyl gallate. J. Plant Physiol. 128, 479-484 (1987). SALEEM, M., A. J. CUTLER, M. COFFEY, and M. LOEWEN: Role of peroxidase in protoplast development: the activity and molecular forms of peroxidase in mitogenic and non-mitogenic protoplasts. In: PUITE, K. J., J. J. M. DONS, H. J. HUiZING, A. J. KOOL, M. KOORNNEEF, and F. A. KRENS (eds.): Progress in Plant Protoplast Research, pp. 137 -138, Kluwer Academic Publishers, Dordrecht (1988). SHARPE, F. T., Jr., A. C. HALL, and G. W. SCHAEFFER: Phosphatidylcholine and phosphatidylethanolamine content of partially synchronized rice cell suspension cultures. Physiol. Plant. 66, 191-196 (1986). SZABADOS, L., Gy. HADLACZKY, and D. DUDITS: Uptake of isolated plant chromosomes by plant protoplasts. Planta 151, 141-145 (1981). TAMIYA, H.: Synchronous cultures of algae. Ann. Rev. Plant Physiol. 17, 1-26 (1966). WANG, H., A. J. CUTLER, and L. C. FOWKE: High frequencies of preprophase bands in soybean protoplast cultures. J. Cell Sci. 92, 575-580 (1989 a). WANG, H., A. J. CUTLER, M. SALEEM, and L. C. FOWKE: Immunocytochemical detection of DNA synthesis in plant cells. J. Plant Physiol. (in press) (1989 b). WEBER, G., E. DE GROOT, and H.-G. SCHWEIGER: Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.). Planta 168, 273-280 (1986).