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Plant Physiol. (1976) 57, 497-503

Synchronous Growth and Plastid Replication in the NaturallyWall-less Alga Olisthodiscus luteus1

Received for publication July 10, 1975 and in revised form November 25, 1975

ROSE ANN CATrOLICO,2 JOHN C. BOOTHROYD,3 AND SARAH P. GIBBSDepartment of Biology, McGill University, Montreal Quebec, Canada H3C 3G1

ABSTRACT

Olisthodiscus luteus is a unicellular biflagellate alga which containsmany small discoidal chloroplasts. This naturally wall-less organism can

be axenically maintained on a defmed nonprecipitating artificial seawa-

ter medium. Sufficient light, the presence of bicarbonate, minimummechanical turbulence, and the addition of vitamin B12 to the culturemedium are important factors in the maintenance of a good growthresponse. Cells can be induced to divide synchronously when subject toa 12-hour light/12-hour dark cyde. The chronology of cell division,DNA synthesis, and plastid replication has been studied during thissynchronous growth cyde. Cell division begins at hour 4 in the dark andterminates at hour 3 in the light, whereas DNA synthesis initiates 3hours prior to cell division and terminates at hour 10 in the dark. Syn-chronous replication of the cell's numerous chloroplasts begins at hour10 in the light and terminates almost 8 hours before cell division is com-pleted. The average number of chloroplasts found in an exponentiallygrowing synchronous culture is rather stringently maintained at 20 to 21plastids per cell, although a large variability in plastid complement(4-50) is observed within individual cells of the population. A change inthe physiological condition of an Olisthodiscus cell may cause an altera-tion of this chloroplast complement. For example, during the lineargrowth period, chloroplast number is reduced to 14 plastids per cell. Inaddition, when Olisthodiscus cells are grown in medium lacking vitaminB12, plastid replication continues in the absence of cell division therebyincreasing the cell's plastid complement significantly.

One of the first successful attempts to induce synchronousgrowth and cell division in a mass population of cells was re-

ported in 1953 for the green alga Chlorella ellipsoidea (27).Since that time, a number of methods have been used to elicit a

synchronous growth response in a variety of algal species includ-ing Euglena (20), Chlamydomonas (1), Scenedesmus (19), Gon-yaulax (26), and Navicula (17). The availability of these systemshas allowed investigators to approach a large range of questionsconcerning the expression and control of specific morphological(2, 4) and biochemical (7, 13) events which take place during a

normal cell cycle.In this communication we report upon conditions which allow

the wall-less marine alga Olisthodiscus luteus to divide synchron-ously when maintained on an artificial seawater medium. Aunique advantage of this unicellular organism is that the many

I Research was supported by the National Research Council of Can-ada (Grant A-2921) and the Quebec Department of Education.

2 Present address: Botany Department, University of Washington,Seattle, Wash. 98195.

' Present address: Department of Molecular Biology, University ofEdinburgh, Edinburgh, Scotland.

chloroplasts found within the Olisthodiscus cell replicate syn-chronously at a time which differs from that of cell division. Inaddition, the chloroplast complement found within an exponen-tially growing cell can be experimentally manipulated. The easewith which chloroplast division and cell division may be uncou-pled in Olisthodiscus makes this alga potentially valuable for thestudy of plastid replication.

MATERIALS AND METHODS

Growth and Maintenance of Cells. Olisthodiscus luteusCarter was obtained from R. R. L. Guillard of the Woods HoleOceanographic Institute, Woods Hole, Mass. The organism wasoriginally isolated by R. J. Conover from Long Island Sound atMilford, Conn. in 1953.

Olisthodiscus was axenically maintained in a defined artificialseawater medium which was developed from a growth medium(14) used for the culture of the red alga Porphyridium cruentum.This modified medium, designated 0-1, contains per liter ofglass-distilled H20: 27 g of NaCl, 6.6 g of MgSO4-7H2O, 5.6 g ofMgCl2 *6H2O, 1.5 g of CaCl2 - 2H2O, 1.0 g of KNO3, 0.07 g ofKH2PO4, 0.04 g of NaHCO3, 1 ml of stock solution A (18.6 g ofNa2EDTA and 2.4 g of FeCl3 6H2O/liter, pH 7), 1 ml of stocksolution B(40 mg of ZnCI2, 600 mg of H3BO3, 15 mg ofCoCl2-6H2O, 40 mg of CuC12-2H2O, 488 mg of MnCl2A4H2O,and 37 mg of (NH4)6MoO24-4H2O per liter), 2.5 ml of 1 M tris-HCl, pH 7.6, and 0.5 ml of vitamin stock solution (0.1 mg ofbiotin, 0.1 mg of B12, and 20 mg of thiamine-HCl made in avolume of 100 ml and stored frozen in small portions in screw-capped test tubes). A second growth medium designated 0-2,which has been routinely used for the growth of Olisthodiscusdiffers from 0-1 medium in that it contains only B12 as a vitaminsource. For heterotrophic growth studies, 0-1 medium was sup-plemented with either beef peptone, glucose, glycerol, mannitol,sodium acetate, or sodium succinate at a concentration range of0, 0.025, 0.05, 0.1, 0.35, and 0.7% (w/v).

Olisthodiscus medium was distributed in 1000-, 500-, or 250-ml volumes to 4-, 2-, or 1-liter cotton-stoppered Pyrex Erlen-meyer flasks, respectively, and autoclaved for 20 min. The finalautoclaved medium contained no precipitate.

Cultures were illuminated from above with General Electriccool white fluorescent lights adjusted to give a light intensity of380 ft-c at the culture surface. The cultures were maintained at19 to 20 C and were gently agitated using a New Brunswickrotary shaker set at 80 rpm. Tests for bacterial contaminationwere made routinely by plating Olisthodiscus culture samples onenriched seawater agar plates and incubating these plates at37 C.

Cell Counts. Haemocytometer: approximately 50 A.l of 5%formalin was added per ml of cells contained in growth medium.These fixed cells were then immediately counted in a Levyhaemocytometer. Less formalin was needed to fix cells whichwere in late linear or stationary phases of growth. These old cells

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CATTOLICO, BOOTHROYD, AND GIBBS

seemed more sensitive than exponentially growing cells andwould easily rupture when the formalin concentration was toohigh. Coulter Counter: Olisthodiscus cells which were suspendedand appropriately diluted in growth medium were counted usinga Model ZB1 Coulter Counter equipped with a 100 ,um aperture.

Chloroplast Counts. Cells were centrifuged at 20 C for 3 minin a clinical table top centrifuge at 2300 rpm. The pelleted cellswere resuspended in growth medium to give a concentration ofapproximately 1 x 106 cells/ml. Five ,ul of this cell suspensionwas placed on a glass slide and covered with a coverslip (22 x 22mm) of 1.5 thickness. With such a small amount of liquid underthe coverslip, the slide starts to dry out almost immediately. Asthis occurs, the individual Olisthodiscus cells swell and ultimatelyburst. When cells have swollen to their maximum and are justabout to burst, all the cell's chloroplasts are spread out in a singleplane of focus and are readily distinguishable from each other.Chloroplast counts were made either at this time or just as thecells burst. Counts were made at 500 x magnification using aZeiss phase contrast microscope.DNA Analysis. A sample containing a total of approximately

1 x 105 cells was axenically removed from the Olisthodiscusculture at 45-min intervals throughout the light/dark cycle. Thecells were pelleted by centrifuging the sample at 2900 rpm in aclinical table top centrifuge at 20 C. All but 0.5 ml of thesupernatant was removed. The cells which were resuspended inthis small volume of medium were then transferred along withtwo 0.2-ml washes of growth medium to a Siliclad-coated testtube (6 x 50 mm). The sample was centrifuged at 7000 rpm at5 C for 8 min in a Sorvall RC2-B centrifuge using an HB-4swinging bucket rotor. The pellet was extracted with 80% ace-tone in H20, and the sample was analyzed for DNA content by arapid filter modification of the Kissane and Robins microfluoro-metric technique (6).

Electron Microscopy. Exponentially growing cells were re-moved from the growth chamber at hr 11.5 in the dark andcollected by centrifugation at 3000 rpm for 5 min at 4 C in aSorvall RC2-B centrifuge. The cells were fixed for 1 hr at 4 C ina fixative containing 5% (v/v) glutaraldehyde, 0.6 M sucrose, and0.1 M phosphate buffer, pH 7.6. The cells were then collected bycentrifugation for 5 min at 20 C at 2900 rpm in a clinical tabletop centrifuge, washed twice at 4 C in 0.2 M phosphate buffer,pH 7.6, and postfixed for 1 hr at 4 C in 1% (w/v) osmiumtetroxide in 0.1 M phosphate buffer, pH 7.6. The cells wererinsed twice at 20 C in distilled H20, dehydrated in a gradedethanol series, and embedded in Spurr's (25) low viscosity epoxyresin. Sections were stained with lead citrate (22) and viewedwith a Philips EM 200 electron microscope.

Reagents. Vitamin B12, D-biotin, and thiamine-HCl were pur-chased from Nutritional Biochemicals Corp. (Cleveland, Ohio).Glutaraldehyde (70%) was purchased from Ladd Research In-dustries (Burlington, Vt.). All other reagents were of analyticalgrade.

RESULTSCell Structure. Olisthodiscus luteus is a unicellular biflagel-

late Chrysophyte alga approximately 12 to 20 ,um long and 8 to14 ,um wide (15). Figure 1 is a representative micrograph of anOlisthodiscus cell grown in 0-1 medium. The cell contains anumber of small peripherally located chloroplasts, which sur-round the large central nucleus. These chloroplasts are approxi-mately 3 to 4 ,um long and 2 to 3 ,um wide. A majority of thechloroplasts are oriented with their pyrenoid region facing thenucleus. Most of the chloroplast DNA is localized in a ring-shaped nucleoid which encircles the rim of the chloroplast justinterior to the peripheral band of thylakoids. In Figure 1, theDNA areas of each chloroplast can be identified because theyare much more electron-translucent than the rest of the chloro-plast matrix. At the low magnification of Figure 1, each chloro-

plast appears to be limited by two membranes. At high magnifi-cation each chloroplast is seen to be completely enclosed by adouble-membraned sac of endoplasmic reticulum as well as by adouble-membraned chloroplast envelope (10). Other cell com-ponents seen in Figure 1 include Golgi bodies, scattered mito-chondria with tubular cristae, a microbody, and various periph-eral vesicles. It is important to note that Olisthodiscus is natu-rally wall-less and is limited only by a single membrane (Fig. 1).This membrane is easily ruptured during fixation and embed-ding.

Ceil Growth. Olisthodiscus cultures double in cell numberevery 25.7 hr during the exponential growth phase when cells aremaintained in 0-2 medium under a continuous light regime. Thisdivision rate occurs after the cultures have passed through ashort lag phase and continues until cells reach a density of 8 x104 cells/ml (Fig. 2). The linear growth phase begins at this cellconcentration and continues until the culture reaches a density of8 x 105 cells/ml.A synchronous pattern of cell division is obtained when Olis-

thodiscus cultures are subject to an alternating 12-hr light/12-hrdark cycle. Cell division begins at hr 3 to 5 in the dark phase ofgrowth and continues until hr 3 to 4 in the light (Fig. 3). Culturesincrease in cell number by a factor of two during each 24-hr cycleperiod. This exponential growth rate is maintained until theculture reaches a cell density of 5 to 7 x 104 cells/ml. Althoughdivision rate significantly declines after this exponential periodof growth is terminated, the final cell concentration which anOlisthodiscus culture may attain is approximately 2.5 x 106 cells/ml. To quantitate the degree of synchrony found in culturesgrown under this light/dark regime, a synchronization index wascalculated according to the following equation (23):

t + T(2 - n)1.12T

where t is the time spent by a culture completing a cell divisionburst, T is the generation time determined for a nonsynchronousexponentially growing culture, and n is 1 plus the fraction of cellsdividing during each division burst. An analysis of 20 synchro-nous cycle experiments has shown that an Olisthodiscus culturecompletes a division burst in 1 1.1 hr (t). The generation time fora nonsynchronous exponentially growing Olisthodiscus culture(T) is known to be 25.7 hr (Fig. 2), and it has been observed thatan Olisthodiscus culture normally makes a 2-fold increase duringeach division sequence (Fig. 3) indicating that every cell in theculture has divided (n = 1 + 1). Given this information, thesynchronization index for an Olisthodiscus culture has beencalculated to be 0.61. Although an ideal culture would have asynchronization index of 1, the value given for Olisthodiscus isquite acceptable when compared to that calculated for othersynchronized systems (1, 9, 23).To determine the amount of DNA present in an Olisthodiscus

cell during different stages of synchronous cell growth, cultureswere sampled every 45 min over a 28-hr period. The amount ofDNA in the culture remains constant until approximately 1 hrinto the dark phase of growth (Fig. 4A). At this time, DNAsynthesis begins and it continues until D-10 (hr 10 in the dark).Cell division (Fig. 4B) initiates at D-4 and does not end until L-3(hr 3 in the light). In this experiment the amount of DNA in theculture increased by 80% during the synchronized cycle ana-lyzed, whereas cell number increased by 86%. Olisthodiscus cellsare extremely fragile, and we believe that the frequent mixing ofthe culture which was necessary during this experiment to ensurehomogeneous sample collection probably is responsible for thefailure of the culture to make a normal 2-fold increase.A schematic diagram of the various phases of the cell cycle of

an Olisthodiscus culture is shown in Figure 5. The G, phaseextends from approximately hr 3 in the light period of growth tohr 1 in the dark when DNA synthesis (S phase) begins. The S

498 Plant Physiol. Vol. 57, 1976

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SYNCHRONOUS GROWTH IN A WALL-LESS ALGA

FIG. 1. Electron micrograph of an exponentially growing cell of Olisthodiscus luteus. The cell has a number of small peripherally locatedchloroplasts surrounding the large central nucleus. cer, chloroplast endoplasmic reticulum; n, nucleoid region of the chloroplast; p, pyrenoid; mb,microbody. x 15,800.

phase continues for about 9 hr and overlaps quite considerablywith cell division (D) which extends from hr 4 in the dark to hr 3in the light. We have not observed a G2 period in the Olisthodis-cus cell cycle but our data does not rule out the possibility thatthere might be a short gap between the end of DNA synthesisand the onset of mitosis. Figure 5 describes the fission harmonyof an entire population of cells rather than the cycle program ofan individual cell.A number of factors affect the efficiency of the growth re-

sponse in Olisthodiscus. For example, cell division rate is quitedependent upon the light intensity at which the culture is main-tained. Synchronous cultures in the exponential growth phase donot make a full 2-fold increase each 24-hr period if the light

available falls much below 380 ft-c. It was also observed thatOlisthodiscus will not survive when grown on a constant darkregime even when the growth medium in which the cells arecultured is supplemented with varying concentrations (0-0.7%)of either beef peptone, glucose, glycerol, mannitol, sodium ace-tate, or sodium succinate. It may be that an insufficient numberof carbon compounds have been tested, but on the basis ofpresent evidence it appears that Olisthodiscus has an obligatelight requirement. Although McLachlan (18) has reported thatthe addition of bicarbonate to artificial seawater medium causedno enhancement in the growth of Olisthodiscus, we have foundthat Olisthodiscus cells cultured in our medium have some de-pendence on this compound for the maintenance of a good

Plant Physiol. Vol. 57, 1976 499



CATTOLICO, BOOTHROYD, AND GIBBS Plant Physiol. Vol. 57, 1976

x

w

C.)

0 5 10 15 20DAY

FIG. 2. Growth of an Olisthodiscus culture maintained on a constantlight regime. Cells were grown on 0-2 medium at a light intensity of 380ft-c. The white bar at the top of the figure indicates that continuousillumination was supplied during culture growth.

x

-J

cj)

-J

w

W._j

0 6 1 2/0 6 1 2p 6 1 2/0 6 1 2P 6 1 2HOUR

FIG. 3. Synchronous growth of an Olisthodiscus culture maintaineden an alternating 12-hr light/12-hr dark cycle. Cells were grown on 0-2medium. During the 12-hr light period cultures received illumination atan intensity of 380 ft-c. White bars at the top of the figure represent thelight phases of growth, black bars represent the dark phases of growth.The numbers to the right of the growth curve indicate the factor by whichcell concentration increased during each cell cycle period.

growth response. Cultures maintained on a L/D cycle in thepresence of varying concentrations of bicarbonate (0-0.38 mM)show an increase in cell division rate such that at the onset of thestationary growth phase, those cultures which had been supple-mented with 0.38 mm bicarbonate contained 24% more cellsthan cultures to which no bicarbonate had been added. It should

be noted that our standard Olisthodiscus media contain 0.48 mmbicarbonate.The presence of vitamins biotin, thiamine, and B12 have been

shown (21) necessary for the maintenance of many marine algalspecies. Olisthodiscus cultures grown in the absence of thesethree vitamins were able to survive for only a short period oftime after which cell death occurred (Fig. 6A). Further studieshave demonstrated that normal cell growth could be maintainedwhen cultures were supplemented with vitamin B12 alonewhereas those Olisthodiscus cells cultured with biotin or thia-

120a)

0

x-J 100

z

80cm

60

4.0

3.5'ob

';3.0x 3

-J

-J

-J

2.0

1.5

HOURFIG. 4. DNA synthesis and cell division during the synchronous

growth of Olisthodiscus luteus. Growth conditions were identical to thosedescribed in Fig. 3. A: DNA synthesis; B: cell number. Each point in thisfigure is a mean of two samples which were obtained from replicatecultures.

L-0

D-6

D-0FIG. 5. Diagramatic representation of the occurrence and duration of

the G0, S, and D periods of the cell cycle of an Olisthodiscus culture.

500

A ~~~~~~~~~~~~~A

A A

AA

A A AA

A*-A

BB

8 12/0 4 8 12/0 4 8 12/0 4

L-6




O 50x

> 40ci3UJ 300

DAYFIG. 6. Effect of the vitamins thiamine, biotin and B12 on the growth

of Olisthodiscus luteus. Cells were maintained on a 12/12 light/darkcycle. Each point represents the mean value of the cell counts made ontwo replicate cultures.

mine as a sole vitamin source did not survive (Fig. 6B). Theaddition of thiamine or biotin did not stimulate the efficiency ofcell growth observed in cultures which contained B12 as the onlyvitamin source (Fig. 6C).

Chloroplast Replication. To determine the chronology of chlo-roplast and cell division during the synchronous growth of Olis-thodiscus, cells were sampled at different stages of the synchro-nous growth cycle and both cell and chloroplast counts were

made. Results of this analysis are presented in Figure 7 which is acomposite of two experiments. As seen in this figure, bothchloroplast and cell number remain constant in the synchronousculture until 2 hr before the onset of the dark period of growth.At this time, although cell number remains constant, chloroplastreplication begins. This increase in plastid number continuesuntil hr 7 of the dark phase of growth. It should be noted thatplastid replication terminates almost 8 hr before cell division iscompleted.A significant degree of variability in chloroplast complement

may be found in a population of Olisthodiscus cells. Cultures inthe exponential growth phase were sampled between L-3 and L-9 of the synchronous growth cycle when it was known thatneither cell division nor chloroplast replication was taking place.A histogram of chloroplast distribution constructed from theanalysis of 1152 cells is presented in Figure 8. These data indi-cate that the average number of chloroplasts found in an Olis-thodiscus cell is 21 and that cells within the population maycontain from 4 to 50 plastids per cell.Although an Olisthodiscus culture rather stringently maintains

this average of 21 chloroplasts per cell during the exponentialgrowth phase, a shift in the mean chloroplast number per celloccurs as a culture progresses through the linear phase ofgrowth. During this growth period, the rate at which the culturedoubles its chloroplast complement does not keep pace with therate at which the culture doubles in cell number. As a result, an

Olisthodiscus culture which contained an average of 21.5 chloro-plasts per cell in the exponential growth phase (Fig. 9A) had amean chloroplast number of 13.9 chloroplasts per cell whensampled in the late linear growth period (Fig. 9B). It has alsobeen observed that specific alterations in the Olisthodiscusgrowth medium may cause a shift in the tightly maintained meanchloroplast number found in an exponentially growing cell. Forexample, if an Olisthodiscus culture is subject to B12 deprivation,cell division will cease for a short period of time before cell deathoccurs. During this interim, plastid replication continues. Thedata presented in Figure 9C represent a culture in which no celldivision has occurred for 2 days. Cells in this culture haveincreased their chloroplast complement 2-fold over that found inan Olisthodiscus culture which was in the exponential phase of

growth. Preliminary experiments indicate that this uncoupling ofcell division and chloroplast replication is reversible by theaddition of B12 to the deficient medium.

DISCUSSION

Olisthodiscus luteus is a naturally wall-less unicellular marinealga which contains approximately 21 small discoidal chloro-plasts. Cells grown on the artificial seawater medium describedin this communication can be induced to divide synchronouslywhen subject to a 12-hr light/12-hr dark regime.The amount of DNA found in an Olisthodiscus cell during the

G, phase of the cell cycle is 2.9 x 10-12 g. This amount is similarto that observed in Euglena (9) and is approximately 10- to 30-fold greater than the cellular DNA content of Chlamydomonas,Chlorella, or Ochromonas (6). Olisthodiscus cells maintained ona 12/12 light/dark cycle replicate their DNA between D-1 and

4.5 ______ 90

4.0 80

HOUR~~~~~~~~~~~~-

3.2.Cou

*. 50 4 8 1A 4R 8 1~4 80 1A

of Olisthodiscus luteus. The figure is a composite of two separate experi-ments. The first experiment was begun at L-4 and was terminated at D-2, while the second was initiated at L-1 1 and terminated at L-6 of thenext cell cycle. Cells were grown in 0-1 medium on an alternating 12/12light/dark cycle.

100

8.0 4.115

1.5 30~~~~~~~~~~~~~22.

0 4 10 15 20 25 30 3520 45 50 5

U-~ ~ ~ ~ ~ HU

FIG. 7. Chloroplat adcliiionduinthenmeofclrpastsnperonucellfoundliof Olisthodiscus lulteuseThenfgr isamp compoingtheotwsexparaenta expri-tnethelasce.Cells weremaintaned mduonan1/1lih/arcyltematng 12eresmlgtdar cyetwe.- n - ftelgh oino h ycrnu

10

20

0 0 1 0 2 0 3 0 4 0 5u~~~O FCLOOLSSCL

FU. 8 itiuini h ubro hlrpat e elfudianOitoicsclueweape0uigteepnnilgotphseCel4eemitie0na1/1 ih/akcceadwr

sampled bestwen-inbL9outelghotion ofthesynrofcoorpatsprchronfousdi

cycle.

501Plant Physiol. Vol. 57, 1976



502

U)-i-i

at

CATTOLICO, BOOTHROYD, AND GIBBS

0 10 20 30 40 50 60 70 80 90NO. OF CHLOROPLASTS/CELL

FIG. 9. Frequency distribution of the number of chloroplasts per cellin Olisthodiscus cultures maintained under different physiological condi-tions. All cultures were grown in 0-2 medium except culture C which was

grown in 0-2 medium lacking vitamin B12. Cultures were grown on a 12/12 light/dark cycle and chloroplast counts were made between hr 5 to 8of the light phase of growth. The number of cells analyzed in asample is expressed as n while x represents the mean number of chloro-plasts per cell found in the culture. A: Exponential growth. Cells were

sampled at a culture density of 4.3 x 104 cells/ml. B: Late linear growthof culture A. Cells were sampled at a culture density of 1.8 x 106 cells/ml. C: B12 deprivation. Cells were sampled at a culture density of 2.9 x

104 cells/ml.

D-10 of the dark phase of growth. The time period between theonset of DNA synthesis and cell division seems to be rathertightly regulated. When cells in the late linear phase of growthare transferred to new growth medium at hr 6 in the light, DNAsynthesis and cell division both commence 3 hr later than they doin a normal exponentially growing culture.

Preliminary experiments have shown that chloroplast DNArepresents approximately 12% of the total DNA in an Olistho-discus cell. Since chloroplast division is limited to a distinct timein the cell cycle, we hoped to also identify a specific period ofchloroplast DNA replication similar to that observed (16) in thesynchronized Chlamydomonas system. No distinct plastid DNAreplication period was observed (Fig. 4). It is possible thatchloroplast DNA synthesis occurs during the same time frame-work as nuclear DNA replication and so is masked by it, or thatour assay is simply not sensitive enough to monitor a small butdefined period of plastid DNA synthesis during the light phase ofgrowth. Alternatively, as suggested for Chlorella (29) and Och-romonas (11), chloroplast DNA replication may occur continu-ously throughout the cell cycle.

It has been observed that chloroplast division and cell divisionoccur as separate synchronous events during the cell cycle ofOlisthodiscus. Although there is a slight overlap (3 hr) in thetime periods of chloroplast and cell division, this is the firstsynchronized system in which plastid replication has been ob-served to begin significantly in advance of cell division. For

Plant Physiol. Vol. 57, 1976

example, in light/dark synchronized cultures of the multiplastidalga Euglena gracilis, the chloroplasts in the cell replicate insynchrony, but this event occurs during the period of cell division(2, 8). In both Olisthodiscus and Euglena, chloroplast divisionoccurs with a tighter synchrony than cell division (2). It would beof interest to know whether all the chloroplasts within a singleOlisthodiscus cell replicate prior to cell division. The fact thatchloroplast division precedes cell division by a relatively longinterval indicates that this may occur, but due to the largevariability in chloroplast number found within an Olisthodiscuspopulation and the overlap which has been observed betweenchloroplast and cell division, it is impossible to be certain. It iswell established for other Chrysophycean algae (24) which haveonly one or two chloroplasts per cell that these plastids replicateprior to cell division.

Analysis of Olisthodiscus cells has indicated that an alterationin the physiological state of the organism resulting from thenormal aging process or from a perturbation in the normalgrowth conditions may be reflected in a change in the chloroplastcomplement of the cell. Cells in the exponential growth phasedisplay a remarkably constant mean distribution of 20 to 21plastids per cell in cycle after cycle even though a large variationin chloroplast number ranging from 4 to 50 chloroplasts per cellis observed in the Olisthodiscus population. During the lineargrowth phase, this stable chloroplast mean is progressively re-duced to 14 plastids per cell. Unlike Olisthodiscus, the numberof chloroplasts in heterotrophically grown Euglena cells main-tained on a continuous light regime increases as this alga moves

through the exponential phase of growth into the stationaryphase (12). We have no explanation at the moment for the modeof chloroplast reduction in Olisthodiscus which occurs during se-

nescence. The fact that a decline in plastid complement is not an

unique occurrence in the Olisthodiscus system is evidenced bythe precise and regulated reduction in chloroplast number whichhas been observed during gametophytic regeneration of themultiplastid bryophyte, Megaceros (3).

It has been shown in this communication that chloroplastdivision and cell division can be uncoupled by starving Olistho-discus cells for vitamin B12. Cells in an advanced state of defi-ciency may contain as high as 3 times their normal plastidcomplement. The effect of B12 deficiency in Olisthodiscus issimilar to that observed in Euglena gracilis (5). It is possible thata degree of control might exist between host cell and plastid inthe regulation of chloroplast division, for in Olisthodiscus thenumber of plastids produced per 24-hr cycle in a B12 deficientcell does not equal the number produced in a cell grown in amedium containing a normal vitamin supply. On the other hand,the absence of B12 may be directly affecting plastid replication.One of the most interesting of the many rhythms expressed

during the synchronous growth of an algal cell is the productionand maintenance of a specific chloroplast complement. Thefunctional efficiency and survival of a cell is dependent upon astrict coordination of the periodicity of organelle biogenesis withcell division. We believe that the synchronized Olisthodiscus cell,in which chloroplast replication and cell division are so easilyuncoupled, may provide an excellent model system to study therelationship between these oscillatory phenomena.

Acknowledgment-We wish to thank D. Liot for his technical assistance in the electronmicroscopy.

LMRATURE CITED

1. BERNSTEIN, E. 1960. Synchronous division in Chlamydomonas moewusii. Science 131: 1528-1529.

2. BOASSON, R. AND S. P. GIBBs. 1973. Chloroplast replication in synchronously dividingEuglna gracilis. Planta 115: 125-134.

3. BuRR, F. A. 1969. Reduction in chloroplast number during gametophyte regeneration in

Megaceros flagellaris. Bryologist 72: 200-209.4. CALVAYRAc, R., R. A. BUrOW, AND M. LErorT-TRAN. 1972. Cyclic replication of DNA and

changes in mitochondrial morphology during the cell cycle of Euglena graciis (Z). EXP.Cell Res. 71: 422-432.

A

12-

n.84

8 -621.5

4L~ I I IL I 1li III I IIIIIIIII III 1II 1I 1 1,B

12

n.87

8 - . 13.9

C4

12

n.73

8 i -46.2

01,1 I I III1 I I III 11 IIilIi I~I.




5. CARELL, E. F. 1969. Studies on chloroplast development and replication in Euglena. 1.Vitamin B,2 and chloroplast replication. J. Cell Biol. 41: 431-440.

6. CATTOLico, R. A. AND S. P. GIBBS. 1975. Rapid filter method for the microfluorometricanalysis of DNA. Anal. Biochem. In press.

7. CATrroLuco, R. A., J. W. SENNER. AND R. F. JONES. 1973. Changes in cytoplasmic andchloroplast ribosomal ribonucleic acid during the cell cycle of Chlanydomonas reinhardtii.Arch. Biochem. Biophys. 156: 58-65.

8. CooKc. J. R. 1966. Studies on chloroplast replication in synchronized Euglena. In: I. L.Cameron and G. M. Padilla, eds., Cell Synchrony. Academic Press, New York. pp. 153-168.

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