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Plant Physiol. (1990) 94, 1568-15740032-0889/90/94/1 568/07/$01 .00/0

Received for publication April 13, 1990Accepted July 12, 1990

Reversible Accumulation of Plant SuspensionCell Cultures in G1 Phase and Subsequent Synchronous

Traverse of the Cell Cycle1

Jerome Conia*, Robert G. Alexander2, Mark E. Wilder, Kimberlynn R. Richards,Mary E. Rice, and Paul J. Jackson

Los Alamos National Laboratory, Genetic Group, Life Sciences Division LS3, Mail Stop M886,Los Alamos, New Mexico 87545

ABSTRACT

The induction of DNA synthesis in Datura innoxia Mill. cellcultures was determined by flow cytometry. A large fraction ofthe total population of cells traversed the cell cycle in synchronywhen exposed to fresh medium. One hour after transfer to freshmedium, 37% of the cells were found in the process of DNAsynthesis. After 24 hours of culture, 66% of the cells had accu-mulated in G2 phase, and underwent cell division simultaneously.Only 10% of the cells remained in Go or G1. Transfer of cells intoa medium, 80% (v/v) of which was conditioned by a sister culturefor 2 days, was adequate to inhibit this simultaneous traverse ofthe cell cycle. A large proportion of dividing cells could bearrested at the Go + G1/S boundary by exposure to 10 millimolarhydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesisby HU was reversible, and when resuspended into fresh culturemedium synchronized cells resumed the cell cycle. Consequently,a large fraction of the cell population could be obtained in the G2phase. However, reversal of G1 arrested cells was not completeand a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA contentwhereas the frequency of cells which remained in Go + G1 phasewas approximately 17%. Incorporation of radioactive precursorsinto DNA and proteins identified a population of nondividing cellswhich represents the fraction of cells in Go. The frequency of cellsentering Go was 11% at each generation. Our results indicate thatalmost 100% of the population of dividing cells synchronouslytraversed the cell cycle following suspension in fresh medium.

Regulation of genetic expression in plants is under thecontrol of interacting processes and involves the biosynthesisof several different classes of molecules. The sequence ofthesereactions is not well known or understood. These regulatorymechanisms are difficult to study using entire plants becauseof the cell heterogeneity within different plant organs. How-ever, the maintenance of uniform plant cells in suspensioncultures will allow these studies. Such cultures are particularlyrelevant when large populations of cells are obtained in thesame, stable biochemical state. Therefore, efforts are required

' This research was conducted under the auspices of the U.S.Department of Energy.

2 Present address: 6 Norway Close, Corby Northants N18 9E9,England.

to induce synchrony of proliferating cells in a specific stage ofthe cell cycle.

Understanding the cell cycle and factors which control it,should provide information about control of these processesin whole plants. Data generated elsewhere with cultivatedplant cells in suspension indicate the repeated passage of thecells through the cell cycle (5, 13, 16). While cytologic obser-vations using microscopy or biochemical measurements pro-vide static analysis of the cell cycle, these methods do notallow continuous monitoring of the cell cycle. As a conse-quence, such analyses limit the conclusions which can bedrawn concerning the effect of different environmental per-turbations upon the cycle. They also do not permit studies ofthe responses to culture modifications beyond one cell cycle.Nevertheless, it has been shown that cell cycle parameters canbe accurately quantified for both plant and mammalian cellsusing image analysis or flow cytometry to measure the nuclearDNA content of single cells within a given population afterstaining with DNA-specific fluorochrome (3, 22, 25, 29).Fluorescence intensities are correlated with the position ofthecorresponding cell within the cell cycle (2, 1 1). Moreover, thesorting capabilities of flow cytometers make possible theisolation of large samples of cells. Such populations can beused to provide an analysis of biochemical properties specificto cells within a specific stage of the cell cycle.

Reported here are the results of investigations carried outto determine the capacity of a population of cultured plantcells in suspension to undergo DNA synthesis after synchro-nization in the first phase (GI) ofthe cell cycle. Accumulationof Datura innoxia cultured cells in GI phase and/or early Sphase was obtained by reversible suppression ofDNA synthe-sis with HU.3 Cell cycle kinetics have been examined bio-chemically and using a single parameter flow microfluoro-meter (20, 30). In vitro cell proliferation can be stimulated orinhibited by chemical agents. However, it would be preferableto conduct studies of the biochemical processes that occur inthe cells without addition of any synchronizing agent. Thesechemicals present potential stresses that could modify theresults. Therefore, techniques have been developed withmammalian cells to induce synchronous traverse of the cellcycle by limiting the amount of an essential growth factor inthe culture medium (27). In this paper, we have developed a

3 Abbreviations: HU, hydroxyurea; CV, coefficient of variation.1568

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SYNCHRONOUS TRAVERSE THROUGH THE PLANT CELL CYCLE

technique in which D. innoxia cells can be accumulated in areversible state ofG,. Large quantities of cells can be synchro-nized in S phase without using any chemical perturbatingagent.

MATERIALS AND METHODS

Maintenance of Datura innoxia Cell Suspension Cultures

The Datura innoxia Mill. cell line was originally obtainedfrom the laboratory of Dr. 0. F. Gamborg (University ofSaskatchewan, Saskatoon, Canada). Suspension cell cultureswere grown in the dark in a modified Gamborg's 1 B5 medium(15) and maintained as already described (19). Suspensionswere transferred as exponentially growing cultures by a four-fold dilution into fresh culture medium preheated to 30°C at2 d intervals. Cell cultures were maintained as one-fifth vol-ume in 250 mL baffled Delong flasks.

Preparation of Conditioned Medium

Two d after subculture, D. innoxia cells were removed fromsuspension by centrifugation at 300g for 2 min. The super-natant (conditioned medium) was collected and passedthrough a 0.2 Mm cellulose nitrate membrane using a 115 mLpresterilized filter unit (Nalgene). The resultant sterile condi-tioned medium could be stored at room temperature forseveral weeks.

Synchronization of Cell Suspension Cultures

Two synchrony-induction techniques were examined. (a)Modification ofthe growth conditions ofthe culture by trans-fer of rapidly dividing, 2 d old cells into a solution containing20% (v/v) new medium and 80% (v/v) conditioned medium.(b) Reversible arrest ofDNA synthesis usingHU (26). Rapidlydividing cell cultures were grown in different concentrations(1, 5, 10, 20, 100 mM) of HU (Sigma Chemical Co.). At 3 hintervals, aliquots of cells were removed from each culture,protoplasts were prepared and nuclei isolated for furtherdetermination of relative DNA content distributions. To re-lease the cells from HU inhibition, the cells were centrifugedat 800g, washed twice, and resuspended into fresh medium.

Protoplast Isolation and Staining

Protoplasts were isolated from suspension cultures by mix-ing an equal volume of cell suspension with a solution con-taining 600 mM KCI, 2 mM NH4NO3, 3 mM CaCl2, 1 mMKH2PO4, 1% (w/v) Cellulysin (Cal-Biochem), and 0.2% (w/v) Rhozyme HP150 (Rohm and Haas, Philadelphia, PA). ThepH of the solution was adjusted to 5.5 with KOH, andsterilized by passage through a 0.2 ,m nylon membrane(Coming) before use. Suspensions were incubated with agita-tion (150 rpm) at 30°C for 90 min. Protoplasts were separatedfrom cells by passage of the digested material through a 30,um nylon sieve. Protoplasts were washed twice in an ice-coldsolution containing one part culture medium and one part ofthe above solution without enzyme. All steps following thesewashes were carried out at 4°C. Protoplasts were then eitherfixed or resuspended in the lysis medium for cell cycle analy-

sis. Fixation was accomplished by passing 3 mL of the aboveprotoplast suspension through a 30 Mum nylon sieve directlyinto 7 mL of ice-cold 95% ethanol. Samples were stored at4°C for at least 1 h prior to further treatment. Nuclei werereleased by resuspending protoplasts in lysis buffer (5 mMHepes [pH 8 with KOH], 50 mM KCI, 10 mM MgSO4) (6).Triton X-100 (Sigma Chemical Co.) was added to a finalconcentration of0.25% (w/v), and the suspension was allowedto set for 10 min at room temperature. Remaining protoplastmembranes were then mechanically disrupted by three re-peated passages of the suspension through a Pasteur pipet.Samples of either fixed or lysed protoplasts were stained withmithramycin (25 Mg/mL, final concentration) and filteredthrough one layer of Miracloth (Calbiochem-Behring Corp.)before being subjected to flow cytometric analysis. Mithra-mycin was a generous gift from Pfizer, Inc. to the NationalFlow Cytometry Resource. Flow cytometric analysis pro-ceeded following exposure to mithramycin for at least 30 minat 4C.

Radioactive Labeling of the Cells

Exponentially growing D. innoxia cells were grown for onegeneration (24 h) in media containing radioactive protein andDNA precursors. Newly synthesized proteins were labeled byaddition of 10 ,Ci/0.1 ,g/mL of [3H]leucine; DNA waslabeled by addition of 10 MCi/I0 ug/mL [3H]thymidine. Allradioactively labeled compounds were purchased from Du-Pont-New England Nuclear, Inc. HU (10 mm final concentra-tion) was added to the cultures for 24 h. Cells were thenreleased from HU inhibition by washing and suspension in aculture medium containing 0.05% (v/v) colchicine (SigmaChemical Co.). Further incubation was for 24 h at 30°C.Radioactively labeled and synchronized cells were then con-verted to protoplasts as described above. Labeled protoplastswere fixed by suspension in a solution containing 25% glacialacetic acid and 75% methanol (v/v) for 24 h.

Flow Cytometric Analysis

The flow analysis of alcohol-fixed protoplasts and isolatednuclei was carried out using the flow microfluorometer (FMF)ofthe National Flow Cytometry Resource (Los Alamos, NM).The beam power of the argon ion laser (Coherent Innova, 90)was 350 mW at 457.9 nm. The yellow fluorescence of theDNA-mithramycin complex was collected through a 512 nmlong wave pass filter.

Cell cycle profiles were analyzed utilizing computer pro-grams supplied by the National Flow Cytometry Resource(1 1).

Sorting of Radioactively Labeled Protoplasts

Radioactively labeled protoplasts were stained with mith-ramycin and analyzed using flow cytometry. Sorting 'win-dows' were set to sort fixed protoplasts according to theirrelative DNA content as determined by fluorescence intensity.Protoplasts in the Go + GI phase and those in the G2 + Mphase of the cell cycle were collected separately. Sorted pro-toplasts were concentrated by subsequent centrifugation at

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Plant Physiol. Vol. 94, 1990

lOOg for 1 min, and washed three times in a 10% trichloroa-cetic acid solution. Suspensions were then placed onto micro-scope slides.

Autoradiography AnalysisSamples of sorted radioactively labeled protoplasts were

applied to microscope slides and squashed by application ofpressure to a cover slip. Cover slips were removed by freezingthe slide on dry ice and dried slides were immersed in Kodaknuclear track emulsion (Kodak Co., Rochester, NY) in totaldarkness. Emulsion was allowed to expose for a minimum of4 d. Slides were then developed in Kodak Microdol-X, washedin running water, then fixed in Kodak Rapid Fixer. Driedslides were stained with a solution containing 50 mm Tris-HCl (pH 7.3), 150 mM NaCl, 30 mM MgCl2, and 100 ,ug/mLmithramycin for 5 min. Slides were then observed using anepifluorescent microscope (Carl Zeiss, West Germany), fittedwith a combination of 450 to 490/FT5 I0/LP520 filters andboth 40x and lOOx neofluar lenses. In each single protoplastthe nucleus was identified according to the fluorescence ofthe DNA-mithramycin complex. The protoplasts were thenscored for the presence or absence of silver grains over theentire cellular content or over only the nucleus.

RESULTSCell Cycle Parameter Analysis

Figure 1 represents the DNA profile ofa 2 d old asynchron-ous population of cultured D. innoxia cells. Figure la corre-sponds to the DNA distribution obtained from a suspensionofisolated nuclei. The left peak has been arbitrarily positionedat channel 80. This peak (CV: 3.6%) corresponds to cells inthe Go and GI phases ofthe cell cycle with a 2C DNA content(35.1% of the total cells). The nuclei in S phase are plottedfrom channel 80 to channel 160. The computer programindicated that 15.2% of the nuclei comes from cells in Sphase. The peak at channel 160 corresponds to cells in G2with a 4C DNA content (frequency: 49.7%), but also includescells in late phases of nuclear DNA replication.

Figure lb illustrates the DNA profile of chemically fixedprotoplasts isolated from the same cell population. Computeranalysis of the cell cycle profiles indicates that 29.0% of thecells are in Go or GI phase; the CV of the corresponding peakis 7.1%. Twenty-one and six-tenths percent ofthe cells appearto be synthesizing DNA (S), and 49.4% are found in the G2+ M phases. The peak corresponding to the cells with a 4CDNA content is usually referred to as 'G2 + M.' This nomen-clature indicates that using single parameter flow cytometryboth the cells in the G2 phase and those in mitosis are classifiedin a composite peak. When analyzing isolated nuclei (Fig. la,Fig. 2, Fig. 3), no mitotic cells would be observed if oneassumes that chromosomes are released individually intosuspension because of the lack of the nuclear membrane.Therefore, in this paper, the peak corresponding to the nucleiwith a 4C DNA content has been referred to as 'G2' unlesscell cycle analysis was performed using fixed protoplasts (Fig.lb) or after colchicine treatment (Table I).

Nondividing Cells (Go)Table I reports the cell cycle parameters of cells cultured in

the presence of radioactive DNA and protein precursors,

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CHANNEL NUMBERFigure 1. Cell cycle analysis of (a) isolated nuclei and (b) alcohol-fixed protoplasts from D. innoxia exponentially growing suspensioncell cultures. Samples were stained with mithramycin and fluores-cence intensities were distributed along a 256 channel linear scale.Two peaks, related to the nuclear DNA content, appeared in thedistribution. 11500 nuclei were counted in each experiment.

followed by sequential 24 h treatments with HU and colchi-cine. Colchicine was added to prevent cells from completingmitosis, and returning from the G2 phase to the G1 phase.Consequently, only nondividing cells (Go) were left in the Go+ GI channels. Only 15.5% of the protoplasts remained inthe Go + G1 phase. Among these protoplasts, only 11%synthesized DNA as determined by the incorporation of [3H]thymidine into nuclei, whereas more than 92% of the proto-plasts contained labeled proteins. The frequency ofprotoplastscontaining labeled proteins was identical for both Go + GIand G2 + M channels. The percentage of protoplasts synthe-sizing both DNA and proteins is approximately 90% amongthe sorted G2-phase protoplasts. These results indicate that aproportion of living cells with a 2C DNA content do notsynthetize DNA. However, a large majority of both dividing

CONIA ET AL.1 570




Figure 2. Suspension of an asynchronous 2 dold culture of exponentially growing D. innoxiacells into fresh culture medium. Cell cycle analy-sis were done at regular intervals for 48 h. Thearrow indicates the population of cells simulta-neously involved in the process of genome rep-lication. Preparation of the isolated nuclei andflow cytometry analysis were as for Figure 1 a.

CHANNEL NUMBER

and nondividing cells are synthesizing proteins. The popula-tion of quiescent cells which were not progressing from 2CDNA content through S to G2 phase and mitosis correspondsto the Go stage. In order to maintain a constant percentage ofcells in Go, some cells from dividing populations must enterGo each generation. The 11% of cells from the Go + GIpopulation which had actively synthesized DNA within theprevious 24 h might represent such cells.

Modification of Growth Conditions

Figure 2 demonstrates the change in distribution of cellsthroughout the cycle in response to transfer into fresh me-

dium. Kinetic events associated with cell activation were

measured after suspension of a 2-d old culture into freshmedium. Aliquots of cells were collected from the culture at2 h intervals. Protoplasts were obtained and nuclei isolated asfor Figure la. Cell cycle analysis revealed that at the time ofsubculture, only a small percentage of nuclei (14.8%) couldbe assigned to the S phase. At t = 0, 35.1% were found in theGo + G, phase, and 50% were in G2. One h followingsuspension in fresh culture medium, a wave of cells simulta-neously began DNA synthesis (arrow). Frequencies of cells ineach phase of the cell cycle determined at regular intervalsthroughout 48 h of culture demonstrate that suspension of 2-d old cells in fresh medium is sufficient to promote DNAsynthesis. After 14 h in fresh culture medium, 10% of thecells were found in Go + GI phase, 21.7% in S, and 68.3% inG2. Later, cells simultaneously underwent cell division andthe population of G2 cells returned to the Go + G1 phase.Increased background values are the result of contaminationof the suspension with chromatin structures from the mitoticnuclei. In the absence of nuclear membrane, chromosomesin different stages of condensation (prophase through telo-phase) are released as isolated chromosomes or chromosomeclumps. The corresponding fluorescence intensities form a

peak at channel numbers below channel 80 (Fig. 2, 36 h).

Flow cytometric analysis of cell cycles revealed the presenceof two distinct populations within the culture (Fig. 2); one

containing twice the DNA content of the other. Therefore,the channels corresponding to the diploid cells in G2 phaseinclude some tetraploid cells in the Go + GI phase. The D.innoxia cell line which were used in these studies exhibited a

low frequency of tetraploid cells. However, when suspendedin fresh medium, tetraploid cells also initiate DNA synthesisas shown by the peak to the right of the G2 peak diploid cells(channel numbers greater than 160). It has not been possibleto determine whether or not the total population of tetraploidcells is constant and established in the suspension cultures, orif some of those cells result from an escape of the mitosis ateach generation of the diploid cells.

Reversibly Arresting Cells in GI Using Hydroxyurea

Figure 3 shows cell cycle distributions at 3 h intervals whenexponentially growing cells were suspended in a condi-tioned:fresh medium (8:2, v/v). Cells were suspended in thesame medium plus HU to induce synchrony. In order toobtain large quantities of cells at the G1/S boundary, cellswere grown for 24 h in media containing 1, 5, 10, 20, or 100mM HU. The efficiency of arresting cells at the Go + G,/Sboundary is dependent upon both the concentration of HUadded and the time of treatment. In Figure 3, data collectedfrom cultures exposed to 10 mm are shown.A final concentration of 1 mM HU did not promote a

significant increase of the cell population in any segment ofthe cell cycle. Arrest at the Go + G,/S boundary was maxi-mized in 9 h of exposure to 5 mm HU. Cells with a 4C DNAcontent increased rapidly thereafter. Following 12 h ofculture,cell divisions occurred and chromatin structures from themitotic nuclei resulted in a broad histogram at channel num-bers lower than 80.

In the presence of 10 mm HU the population of cells which

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Figure 3. Upper row of each panel: DNA distribution patterns of anexponentially growing D. innoxia cell culture following suspension in80% conditioned medium. Cell cycle distributions are redundant fromt = 0 h to t = 24 h of culture. Lower row: DNA distribution patternsobtained from cells grown in the presence of 10 mm hydroxyurea.After 24 h of culture suspension of cells into fresh medium inducedsynchronous traverse of the cell cycle. The arrow indicates thedispersion of the chromosomes. Preparation of the isolated nucleiand flow cytometry analysis were as for Figure 1 a and Figure 2.

initiate genome replication moves from the Go + GI phase tothe G2 phase much slower than the controls. A maximum ofcells arrested at the Go + G,/S boundary was obtained after12 h of treatment using 10 mM HU (Fig. 4). The frequencyof cells in the G2 phase decreased until 12 h, then remainedstable since no more cells underwent cell division. After 12 h,the cells progressed through the S phase (Fig. 3, Fig. 4).Therefore, after 24 h a large proportion of the cells were inGI, S, and late S phases. However, no increase in the numberof G2 cells was observed. By comparison, the cell cycle distri-butions obtained from cells growing in the absence ofHU are

very consistent and reproducible throughout the entire exper-iment. The average percentage of cells in Go + GI was 48.4± 5%, cells in S represented 19.1 ± 8%, and cells in G2

represented 32.5 ± 5% of the entire population (Fig. 3,control).

Cells exposed to 20 mM HU responded similar to cellsgrowing in 10 mM HU. However, cells in late S phase wereobserved after 15 h of culture instead of 18 h. When using100 mm HU, cell cycle analysis was reliable to 12 h, amaximum of cells in G1 appeared from 9 to 12 h. After 12 hof culture, the cell cycle parameters and segments could notbe determined with confidence, probably due to a loss ofculture viability.

Traverse of Synchronized Cells Through the Cycle

The preceding results indicate that a large number of cellscan be synchronized at the Go + G,/S phase boundary bycombining growth in conditioned medium and the use ofHU. Such a cell population is suitable for studying the effi-ciency of simultaneous DNA synthesis after release from HUinhibition. Once washed and suspended in fresh medium,cells that had accumulated in GI and early S phases completedDNA synthesis. Consequently, a large number of cells accu-mulated in G2, a fraction of which simultaneously beginmitosis, and return to G,3. Figure 3 shows an increase ofeventsper channel in channels 0 to 80 (arrows) 3 h after washing thesynchronized cells. This peak results from the dispersion ofthe chromosomes and is larger 14 h after washing the HU-treated cells, indicating that the number of cells undergoingmitotic division increased significantly. At this time a largepercentage of cells are in G2 (73.9%), whereas only 17.2%remain in the G0+ GI, and 8.8% in S.

DISCUSSION

If flow cytometry is to be applied to the study of the cellcycle, samples, either isolated nuclei or fixed protoplasts,should correspond to a true representation of the culturedcells themselves. Cell cycle analysis obtained from both iso-

Table I. Frequency of Cells Incorporating DNA and ProteinRadioactive Precursors after Successive Incubations intoHydroxyurea and Colchicine

Exponentially growing D. innoxia Mill. cells were grown in thepresence of either a DNA or a protein precursor. Hydroxyurea (10mm final concentration) was added to the cultures for 24 h. The cellswere then washed and resuspended in a culture medium containing0.05% (w/v) colchicine for 24 h. Protoplasts were prepared by en-zymic digestion of the cell wall, washed in ice-cold medium and fixedin acetic acid:methanol (1:3, v/v). Fixed protoplasts were stained withmithramycin and sorted using flow cytometry according to theirrelative DNA content. Protoplasts from both Go + G1 and G2 + Mpeaks were purified and placed on a microscope and the slide wascoated with photographic emulsion. After 14 d, autoradiographs weredeveloped and silver grains were scored for their nuclear or cyto-plasmic location in 1300 sorted protoplasts.

Cell Cycle Precursor IncorporationaDistributions [3Hthymidine rHileuidne

Go + G, 15.5 10.9 92.4G2 + M 62.9 89.4 93.5

a Results given in %.

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Time (hours)Figure 4. D. innoxia cell cultures grown in the presence of 10 mmhydroxyurea for 24 h. Frequencies were obtained after the DNAdistribution patterns shown in Figure 3. (E3), Go + G,; (A), S; (0), G2phase. After 24 h of culture, cell suspensions were washed twiceand resuspended in fresh culture medium (arrow). Synchronoustraverse of the cell cycle resulted in a rapid increase in the number ofG2 cells and a decrease in the number of cells in S.

lated nuclei and fixed protoplasts of D. innoxia producedsimilar cell cycle phase ratios. However, resolution of theDNA content distributions obtained from isolated nuclei weresignificantly better (CV: 3.6%, Fig. la) than those obtainedfrom fixed protoplasts (CV: 7.1%, Fig. lb). Boundaries be-tween S and Go + G, and G2 phases were more pronounced,and an overestimation of the percentage of cells in S occurredusing fixed protoplasts. Therefore, isolated nuclei appearedmore suitable for studying synchronized populations of Da-tura cells. The use ofisolated nuclei-reduces variability causedby the presence of plastid DNA and nonspecific staining ofmembranes. However, analysis of isolated plant nuclei do notprovide a means of studying and sorting intact cells fromdifferent stages of the cell cycle.To avoid any loss ofDNA, the process of protoplast release

was monitored using an inverted microscope. Time of diges-tion was as short as necessary to convert approximately 90%of the cells to protoplasts. These times were consistent (90min at 30°C) for all data points. The stability of the isolatednuclei was also tested. Suspensions could be stored afterstaining for at least week at 4°C with no loss of resolution(data not shown).The area between the Go + GI and G2 peaks corresponds

to the cells in different stages of S phase (Fig. la). Cells withfluorescence intensities close to Go + GI peak are in earlierstages of genome replication than cells with fluorescenceintensities close to the G2 peak. The cell cycle analysis com-puter program at Los Alamos National Laboratory ( 11) cor-rects for the overlap of S phase cells into both the Go + G1,and G2 peaks. Consequently, this program not only allowsthe discrimination between the Go + G1 and the G2 cells but

also permits an accurate measurement ofthe frequency of theS phase. With samples containing a high level ofbackground,the number of cells in S phase may be overestimated bycomputer analysis (1). Computer facilities are available forprecise analysis of the cell cycle parameters. Adequate meth-ods should produce accurate and consistent measurementsnot only with data generated from asynchronous cell popu-lations (when cells are equally distributed throughout the cellcycle), but also when a given population of cells predominateas in synchronously growing cell suspension cultures.

Single parameter flow cytometry proved useful to establishthe passage of cultured plant cells through the S phase withtime. A large number of experiments using plant cell lines ofD. innoxia, Petunia hybrida, Lycopersicon esculentum, andZea mays (our unpublished results) confirm the reproduci-bility of these experiments. We have also obtained consistentmeasurements by determining the cell cycle kinetics of the D.innoxia suspension cell cultures using methods which havebeen extensively described by others (5, 16). Those methodslead to the determination of the growth rate by cell countingafter protoplast production, by measurement of turbidity andpacked volume of cells, and by the radioactive labeling of thecells. The usefulness of flow analysis to study cell cycle distri-butions of plant protoplasts has already been demonstratedfor several other plant species (7, 18, 24), and have beenreviewed (4, 14). Single parameter flow cytometry has theadvantage of simplicity and reliability, making series of inves-tigations possible over extended periods oftime. Biparametricanalysis using two sources of fluorescence may provide moreinformation. However, this kind of flow cytometry is moreexpensive and the preparation of the samples is significantlymore complex. High resolution cell cycle studies could beobtained by combining DNA with RNA measurements (9,28). Moreover, immunocytochemical techniques have beencombined with flow cytometry to allow the distinction be-tween replicating and nonreplicating cells (10, 17). For ex-ample, in mammalian research and more recently in plantsciences, dual parameter flow cytometry has been successfullyapplied to cell cycle analysis after incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the genome during DNA repli-cation (12, 21, 23).

Modification of growth medium conditions was effectivefor accumulating plant cells from different plant species at aspecific point in the cell cycle. Synchronization is usuallyrelated to the addition or removal of a particular growthregulator (8). The reduction in the number of cell divisionsin D. innoxia cell cultures suspended in 2-d old conditionedmedium can result from either the consumption ofone crucialgrowth factor or the generation ofan inhibitor synthesized bythe cells. Addition offresh media results in further cell divisionsuggesting that the former is most likely.

Suspension cell cultures ofD. innoxia are reversibly arrestedat the GI/S boundary of the cell cycle after inhibition of theDNA synthesis with 10 mm HU (Fig. 3). It has been shownin mammalian cultures that HU reduces the rate of cellprogression from G, to S, preventing initiation of DNAreplication (27). D. innoxia cells were also suspended in a80% conditioned medium containing HU to avoid the freshmedium-dependent activation of the DNA synthesis. Conse-quent exposure time to HU could be minimized, avoiding

1 573




cytotoxicity effects of HU upon non-S phase cells (27). Syn-chronous cell cycle traverse of the D. innoxia cells both afterHU treatment and after suspension in fresh culture medium,involved most of the population of dividing cells. However,cell cycle distributions (Figs. 2 and 3) revealed some cellsremaining in the Go + G1 phase (typically 10-17%). Studiesincorporating radiolabeled precursors of DNA and proteins(Table I) demonstrated the presence of a fraction of culturedcells (frequency about 11%) which are not actively dividingbut are still metabolically active.

Preliminary data demonstrates a maintenance of a syn-chronous population of dividing cells 2 d after release fromHU inhibition (our manuscript in preparation). Therefore,such a cell suspension should be useful for the study ofperiodic biochemical events or synthesis of macromoleculesand regulatory processes related to specific stages of the cellcycle. The ability of D. innoxia cell suspensions to respond toperturbating agents ofthe cycle, and the ability to synchronizecultures with fresh culture medium, with no need of chemicalagent, makes this cell line a good model for further investi-gations. Such a cell culture should provide information re-

garding cellular biochemistry and molecular synthesis relatedto the cell regulation and should allow the development of a

better understanding of mitotic processes in plant cells.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Robert A. Tobey for useful discus-sions and critical comments. We gratefully acknowledge CherylKuske and Valerie Sisneros for careful reading of the manuscript.

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