A microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox

DEVELOPMENTAL

BIOLOGY

94, ,441-450 (1982)

A Microspectrofluorometric Analysis of Nuclear and Chloroplast DNA in Volvox ANNETTE Division

W.~OLEMANAND

MARKJ.MAGUIRE

of Biology and Medicine, Brown University, Received March

Providence,

Rhode Island 02912

9, 1982; accepted in revised form July 30, 1982

Nuclear division immediately follows nuclear DNA doubling in all stages of the life cycle examined in the green alga Voluox; fluorescence microfluorometry of individual cells revealed no evidence of prolonged accumulation of nuclear DNA prior to mitosis in reproductive cells. Somatic cell nuclear DNA quantity is unaffected by developmental events in gonidia of the same spheroid; it remains constant from the end of cleavage until the death of the cell. In reproductive cells, chloroplast DNA replication precedes nuclear replication. The sites of plastid DNA accumulation, made visible by use of the fluorochrome 4’,6-diamidino-2-phenylindole, increase in number during the prolonged growth phase of the V. carteri gonidium. Microspectrofluorometry of fluorochrome-stained DNA in situ shows that plastid DNA increases exponentially throughout this phase. The continuous plastid DNA accumulation during gonidial growth appears to represent a prokaryote-like instead of a eukaryote-like control of DNA synthesis. Most somatic cells contain plastid DNA, and this d’oes not increase in amount during colony growth and reproduction. Most sperm cells also contain plastid DNA, although approximately 5% of somatic cells and up to 20% of sperm cells have no discernable plastid DNA. This is the second group of organisms in which DNA-free plastids have been observed. INTRODUCTION

The colonial green flagellate, Voluox, has become increasingly a focus of studies on morphogenesis, not only for its peculiar morphogenetic pattern in vegetative spheroid formation but also for the study of genetic control of development, and for the analysis of the hormonal substances which trigger sexual reproduction (Huskey and Callahan, 1980; Gilles et al., 1981; Starr et al., 1980). Yet the nature of its basic cell cycle has remained unclear. This results primarily from the difficulties associated with obtaining and satisfactorily analyzing the DNA from suitably synchronized cells of different types and starges. An alternative approach is provided by microspectrophotometry. This methodology permits the measurement of DNA quantity in individual nuclei without requiring any complex prior manipulation of the cells. Although the haploid Voluox nucleus contains only approximately 2 X lo-l3 g of DNA (Kochert, 1975; Margolis-Kazan and Blamire, 1976), this small amount can be measured easily after staining the cells with the fluorochrome 4’,6-diamid.ino-2-phenylindole (DAPI),l a compound which shows enhanced fluorescence specifically when bound to double-stranded DNA. The amount of fluorescence shown by the DAPI-DNA complex is proportional to the amount of DNA (Coleman et al., 1981). Measurements are directly comparable as long ’ Abbreviations used: DAPI = 4’,6-diamidino-2-phenylindole; = chloroplast DNA.

as the DNA is of the same base composition, for DAPIDNA fluorescence is influenced by the A-T content of the DNA (Lin et al., 1977). The present paper presents the results of a microspectrofluorometric analysis of both nuclear and chloroplast DNA in the various cell types and developmental stages of the life cycle of Voluox. The fluorochrome used is DAPI, although the same experiments have been repeated using mithramycin, with the same results. These results define the nuclear cell cycle as similar to that of other Volvocalean organisms, and present for the first time the nature of the chloroplast DNA cycle in a eukaryote cell, as measured directly on single cells. Preliminary data on the nuclear cell cycle as measured by Feulgen microspectrophotometry have been published earlier (Coleman, 1979b). MATERIALS

AND

METHODS

Clones of two species of Volvox have been examined, all obtained from the Texas Culture Collection of Algae: (Starr, 1978) V. aureus UTEX No. 1899 and V. carteri UTEX No. 1885 (P), 1886 (a), and clone 70-52. All were maintained at 24°C in a culture chamber having a 1ight:dark cycle of 16:8 hr per day. Approximately 80 g einsteins/cm/sec of light were provided by a bank of fluorescent lamps supplemented with incandescent bulbs. Stocks were maintained, and sometimes sampled for measurement, in soil water cultures, but the data presented were obtained from cultures growing in a chem-

ctDNA

441 0012-1606/82/120441-10$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in e.ny form reserved.

442

DEVELOPMENTAL BIOLOGY

ically defined nitrate and Blz-containing medium, VolVOX medium (Starr, 1978). Spheroids (colonies) and cells were concentrated by gentle centrifugation and a small drop was placed on a gelatin-coated slide. A coverslip was put in place and the preparation gently squashed to flatten but not break the cells. The slide was then immediately placed in liquid nitrogen, and after freezing, the coverslip was removed and the slide fixed in ethanol:glacial acetic acid (3:l) for an hour at room temperature. Subsequently, slides were dehydrated through an alcohol series, allowed to air dry, and stored for later examination. For staining, slides were first presoaked in 200 mA4 KCl, a step which helped to reduce any nonspecific stain binding. Twenty-five microliters of 0.1 pg/ml DAPI in McIlvaine’s pH 4 buffer (Coleman, 1978) was added to the blotted slide and a coverslip put in place. After 3060 min the preparation was pressed gently with blotting paper and the coverslip ringed with clear nail polish to retard drying. Observations were made within l-2 days of slide preparation, during which time no detectable change in fluorescence values occurred (Coleman et al., 1981). Prior to staining, some slides were pretreated with heated pancreatic RNase and others with calf thymus DNase as described by Coleman et al. (1981) in order to evaluate the specificity of the staining. To ascertain whether the fixation treatment detectably altered the appearance of cell DNA, living colonies were suspended in a solution of 0.5 pg/ml DAPI in distilled water and examined with the fluorescence microscope after several hours. Such colonies after subsequent rinsing remained fully viable and could be used to establish fresh cultures. Stained preparations were examined with a Zeiss photoscope capable of phase microscopy and also fitted with a 100-W mercury lamp arranged in the epifluorescence mode for fluorescence observations. For DAPI the 48 77 02 filter combination was used, which provided 380nm light for excitation and passed the 460- to 480-nm fluorescence emitted to the eye. Measurements of fluorescence were made by inserting the appropriately sized field diaphragm and measuring pinholes in the light path, and aligning the object of interest in the center of the pinholes. The spectrophotometer was then activated and the reading recorded as previously described (Coleman et al., 1981). Where significant background fluorescence was observed adjacent to the measured object, a measurement was also made on this area and subtracted from the value for the object of study to obtain a corrected reading. The uniformity of staining throughout a slide was evaluated by adding an aliquot of chick red blood cells to the dry slides before staining, and then measuring scattered red blood cell nuclei to assess their uniformity (Coleman, 1981). There

VOLUME 94. 1982

appeared to be no significant sponse.

variability

in staining

re-

RESULTS

Volvox is a haplont, with only one diploid cell in its life cycle, the zygote (Starr, 1975). The vegetative spheroid consists of two easily distinguishable types of cells, large gonidia which are capable of forming new individuals, and small somatic cells which die after a period of several days (Fig. 1). The gonidia of V. carteri enlarge steadily, after release of the young spheroid, until they reach a diameter of approximately 75 pm. At this point they undergo a rapid series of cell cleavages to produce an entire daughter colony containing approximately 2000 cells. During the gonidial growth period, its nucleus also enlarges and becomes even more faintly staining. The massive size of the nucleus in a mature gonidium, along with initial biochemical studies suggesting a parallel DNA accumulation (Margolis-Kazan and Blamire, 1976), would imply that the mature Volvox carteri gonidial cell nucleus contains multiple nuclear genomes. On the other hand, nuclear volumes are known to alter with metabolic state, without comparable DNA increase, as seen in developing oocytes and in the increase of red blood cell nuclear volume as it is reactivated subsequent to cell fusion with a cycling cell. Direct measurement of the DNA per Volvox nucleus at the time of division should distinguish between these two explanations of the nuclear volume increase. Nuclear DNA Cleavage

Values during Gonidial

Growth and

As shown in the data in Table 1, there is no evidence for the occurrence of nuclear polyploidy in the vegetative cycle of V. carteri. No nucleus had even the 2c amount of DNA, as judged by the fluorescence of the lc nuclei in adjacent somatic cells, although one example approached this value. Instead the interphase nuclei of the rapidly cleaving embryo appear to be almost constantly in S phase. The nucleus of the fully enlarged gonidium just prior to the cell rearrangements preparatory for cleavage is impossible to measure without including a small component of plastid DAPI-DNA fluorescence. Hence the fluorescence values for gonidial nuclei in Table 1 include a contribution of plastid DNA. Even so, they would appear to maintain only the lc level of nuclear DNA throughout their prolonged growth period. Comparable results were obtained for developing embryos of V. aureus although the distribution of plastid DNA affected both gonidial and embryonic nuclear measurements (Fig. 2). In this species, the first cleavage occurs when the gonidium is small, with further cleav-

COLEMAN AND MAGUIRE

Volvox

Nuclear

and

Plastid

DNA

Voluox

443

Cycle

TABLE 1 NUCLEAR DNA

carteri

Fluorescence Cell type Somatic Sperm Mature gonidium Binucleate embryo Quadrinucleate embryo

per nucleus

N

Mean

Range

C.“.

20 20

101.30

76-145 73-120

17.5 13.5

93.50

5 6

146.0 133.2

8

134.2

116-178

18.4

116-164

16.8

70-192

21.3

Note. Fluorescence of individual nuclei was measured in DAPIstained preparations of V. carteri. N = sample size, C.V. = coefficient of variation.

somatic cells, those in spheroids where embryo development had been completed (Table 2). Hence control of nuclear DNA synthesis in developing embryos is entirely separate from that in the adjacent somatic cells. Somatic cells have arrested in G1 of the nuclear cycle. In the male clone of V. carteri, the nuclear fluorescence in sperm nuclei was also measured, and conformed to the expected lc value (Table 1). Chloroplast

DNA in Vegetative Reproduction

A Voluox cell has only one, cup-shaped chloroplast. Chloroplast DNA (ctDNA) is easily seen in the somatic cell plastid of Voluox (Fig. 3), and can be quantitated

FIG. 1. Phase and fluorescence micrographs of a very young gonidium of V. aureus stained with DAPI. In the gonidium, the relatively large nucleus is discernable as well as more than 50 ctDNA nucleoids. Somatic cells, with their nuclei and ctDNA nucleoids, surround and overlie the gonidium. x1700.

ages alternating with periods of growth of the embryo, rather than being delayed until the end of the gonidial growth phase (Darden, 1966). No difference in nuclear DNA fluorescence values was found between “young” somatic cells, those in spheroids where the gonidia had not yet begun division, and “old”

FIG. 2. Fluorescence micrographs of V. aureus gonidium at the eightnucleate stage of cleavage, stained with DAPI. In addition to the eightnuclei, numerous ctDNA nucleoids are visible. X1700.

444

DEVELOPMENTAL BIOLOGY TABLE 2 VO~UOXQUWUSSOMATICCELLNUCLEAR DNA Fluorescence

VOLUME 94, 1982

a 20 Lk-

per nucleus

N

Mean

Range

C.“.

Parental colony Daughter colony

10 10

119.5 117.8

1033141 78-137

10.4 16.6

Parental colony Daughter colony

10 10

101.5

79-128

17.5

87.2

71-111

16.0

Parental colony Daughter colony

10 10

108.8 119.7

72-135

16.6

96-150

16.5

e

0 f b 20 0 LL 0

4

8

Note. The fluorescence of DAPI-stained nuclei was measured in parental somatic cells and daughter somatic cells of three spheroids. N = sample size, C.V. = coefficient of variation.

by microspectrofluorometry. As with Chlamydomonas and with other members of the colonial green flagellates (Coleman, 1978), the ctDNA is seen as condensations, “nucleoids”, scattered through the chloroplast. These are irregular in shape and size, and may be interconnected in some fashion beyond the resolution of this methodology. Living cells exposed to the dye have the same appearance.

NUMBEROF CTDNA NUCLEOIDSPER PLASTID FIG. 4. Effect of spheroid age on the number of chloroplast DNA nucleoids per somatic cell chloroplast in V. carteri. Actively growing colonies were fixed and stained with DAPI, and the number of DNA nucleoids counted in each somatic cell chloroplast, following a random path within a colony. a = 100 somatic cells in a colony containing gonidia 16-18 pm in diameter. b = 100 somatic cells in a colony containing gonidia 33 pm in diameter. c = 100 somatic cells in a colony containing gonidia 33 pm in diameter. d = 100 somatic cells in a colony containing gonidia 50 pm in diameter. e = 100 somatic cells in a colony containing gonidia 75-90 pm in diameter. f = 100 somatic cells in a colony containing gonidia which had finished cleavage. g = composite of 1000 cells from spheroids at all stages of development. Thirty-seven of the thousand somatic cells had no detectable chloroplast DNA nucleoids. Abscissa = number of nucleoids per plastid; ordinate = number of cells (= number of plastids, since there is one plastid per cell in volvox.)

Nucleoid Number per Vegetative Cell

FIG. 3. Fluorescence micrograph of a portion of a V. carteri spheroid displaying individual somatic cells. One of these (arrow) appeared to have no ctDNA when carefully examined at various focal planes. There was no fluorescent particle associated with its plastid region, and the bright spot associated with its nucleus is a condensation typical of all somatic cell nuclei in this species. X1700.

Under conditions of normal vegetative growth in VolVOXmedium there is a mean of 5.3 nucleoids per somatic cell in V. aureus, and 2.3 in V. carteri. As shown in Fig. 4, there seems to be little change in nucleoid number per somatic cell during the aging of a spheroid. The observed small increase in the mean number of nucleoids per cell (Fig. 4: a = 1.5; b = 1.6; c = 2.2; d = 2.9;

COLEMAN AND MACUIRE

Volvor

e = 2.4; f = 3.4) may only reflect the increased ease with which neighboring nucleoids can be distinguished as the somatic cells enlarge dramatically. Microspectrofluorometric measurements of the plastid nucleoid region of young versus aged somatic cells, although quite variable due to the nonspecific binding of the dye to extracellular materials, fail to reveal any significant increase in ctDNA fluorescence per cell. For example, mean ctDNA fluorescence per grandmother somatic cell = 29.6 units while mean ctDNA fluorescence per mother somatic cell = 39.4 units. Somatic cells of the daughter were too tightly apposed to permit such measurements. Hence the control of ctDNA synthesis in somatic cells is entirely separate from that of gonidia, just as is true for their nuclei. A small proportion (ca. 5%) of somatic cells at all stages of colony development examined appeared to lack any ctDNA (Figs. 3 and 4). This was true of both V. aureus and V. carteri. 13y contrast, only about one somatic cell in a thousan’d of these two species was anu-

Nuclear

DNA

445

Cycle

TABLE 3 Voluox carteri f. NACARIENSIS SOMATIC CELL DNA Nuclear fluorescence Number of nuclei Mean fluorescence Range

value per nucleus

Cytoplasmic fluorescence value Number of cells Mean fluorescence in chloroplast Range

25 281.5 260-318 25 15.5 9-25

Note. DAPI-stained somatic cells were used to measure the fluorescence of individual nuclei and also the fluorescence associated with the chloroplast in the same cell, using the same small pinhole, magnification, and settings for all readings.

cleate or binucleate, attesting to the efficacy of the spindle apparatus in ensuring successful bipartition at cleavage. Clearly, the vast majority of somatic cells receive ctDNA, so that their inevitable death without issue cannot be attributed to a lack of ctDNA per se. Chloroplast DNA in gonidia is also found as nucleoids. The gonidia of Volvox contain an obviously enhanced amount of ctDNA over that in adjacent somatic cells from the earliest time at which their increased size reveals their gonidial nature. This is true of all the clones examined, and is illustrated for V. aureus and V. carteri in Figs. 1 and 5. The number of ctDNA nucleoids increases continuously from the time of the inception of gonidial enlargement. Their accumulation overlaps with the nuclear divisions which occur regularly in V. aureus daughter colony formation, while proceeding in the total absence of nuclear DNA synthesis and division in V. carteri. In this latter species, nuclear divisions are postponed until the end of the prolonged gonidial enlargement stage. In fully enlarged gonidia of V. carteri the quantity of ctDNA is so great that it is difficult to obtain fluorescence photographs showing the nucleus. Nuclear

FIG. 5. Phase and Auoresce:nce micrographs of various sizes of DAPIstained gonidia of V. carteri 70-52, illustrating ctDNA nucleoids. Smallest gonidium = 12 pm; largest = 47 pm in diameter.

and Plastid

Versus Plastid DNA Quantitation

The results of an attempt at relative quantitation of somatic cell nuclear and ctDNA are presented in Table 3, where a small pinhole was used to measure selectively the nucleus and then the cytoplasmic region containing all or most of the ctDNA nucleoids. These data suggest that 5-10% of the DNA in the average somatic cell represents DNA in the chloroplast, a value comparable to those obtained by biochemical methods for V. carteri and for Chlamydomonas reinhardi (Kochert, 1975; Howell and Walker, 1972; Chiang and Sueoka, 1967). However, since the nuclear and plastid DNA differ in base composition, their values obtained by DAPI-DNA fluorometry cannot be directly compared without first obtaining the correction factor resulting from their differing base compositions.

446

DEVELOPMENTAL BIOLOGY

VOLUME 94, 1982

Hence, the ctDNA cell cycle displays continual DNA synthesis, more like that found in the cell cycle of prokaryotes than that of eukaryote nuclear DNA. Sexual Reproduction

FIG. 6. Fluorescence micrograph of DAPI-stained gonidium of V. carteri 70-52 at stage approaching cleavage. Diameter = 74 pm.

Quantitation

of Gonidial ctDNA

during Development

Direct comparison can be made, however, of ctDNA values at different stages of cell development. In order to quantitat,e the accumulation of ctDNA during gonidial development, the 70-52 mutant of V. carteri 1885 was utilized, which bears the mutant locus “dissociator” (Starr, personal communication). This mutant lacks the enclosing sheaths which hold the spheroid together; hence the cells fall apart shortly after inversion of the embryo. Such a culture contains free gonidia at all stages of enlargement and cleavage, as well as somatic cells, and can be treated as a preparation of unicells (Figs. 5 and 6). Since full grown gonidia are so large, a relatively large pinhole and 25X objective were used to measure a random sample of gonidia, although a few of the very largest could not be included. Under this magnification, the nucleus of the enlarged uncleaved gonidium is so faint that it cannot usually be distinguished, even by fluerescence. However, somatic cell nuclei remain condensed. With these measurement conditions they emit 2-3 units of fluorescence (log, 2.5 = 0.9), so that the nuclear contribution to gonidia DAPI-DNA fluorescence is negligible. Such small cells were not included in the graph because most appear to be somatic rather than gonidial. The diameter of all gonidial cells was also measured. Since the method of slide preparation results in very flattened spheroids, discs of uniform thickness, the assumption was made that gonidial volume is proportional to gonidial area. The data obtained (Fig. 7) indicate that, despite some degree of scatter, the DAPIDNA fluorescence increases exponentially as a function of the increase in cell volume. The regression obtained, log, (fluorescence) = a + b log, (gonidial area), is significant at the level of P < 0.001 (r2 = 0.77). There is no indication that ctDNA increase is restricted to any one period within the prolonged gonidial enlargement phase.

In V. aureus, spheroids of a single clone form either male or facultative female spheroids at the time of sexual reproduction (Darden, 1966). The male colonies can be recognized by the fact that nearly half of the cells enlarge and become androgonidia, each forming a sperm packet containing 32 or 64 sperm cells. The size of these androgonidia at division is approximately the same as that of vegetative gonidia at their first division, but androgonidia continue to cleave rapidly with little or no further increase in overall size evident in the completed sperm packet. At cleavage, androgonidia contain numerous ctDNA nucleoids, comparable to what is seen in vegetative gonidia at the same size. By the 32-nucleate stage, where the majority of the androgonidia ceased cleavage and began final differentiation, an estimate of the number of cpDNA nucleoids per sperm packet was made. The number varied widely from packet to packet, ranging from 18 to 150. On the average, the number of nucleoids is sufficient to provide about one nucleoid per sperm cell. Examination of mature sperm reveals that this is generally the case. However, there appears to be considerable variability, and approximately 20% of sperm have no discernable ctDNA under our growth conditions. In the male spheroids of V. carteri, the androgonidia undergo rapid cleavages, forming sperm packets, when about the same size as cleaving asexual gonidia or an-

loge

gonidial

area

FIG. 7. Accumulation of plastid DNA in Voluox gonidia preparing for asexual reproduction. Gonidia of V. carteri 70-52 at various stages of enlargement were fixed and stained with DAPI. Both the diameter and the DAPI-DNA fluorescence of 73 gonidia were measured. Abscissa = gonidial area (see text) = log, T? Ordinate = log, fluorescence units.

COLEMAN AND MAGUIRE

Voloox

Nuclear

and Plastid

DNA

Cycle

drogonidia in V. aureus (Starr, 1971). The 64-nucleate sperm packet and the released sperm display numerous ctDNA nucleoids (Figs. 8 and 9). Again, as in V. aureus sperm, there appears to be considerable variability in the number of ctDNA nucleoids per sperm cell. For 85 sperm counted in one h.arvest there was a mean of 2.6 nucleoids per sperm cell, with a range of O-6, and 14% appeared devoid of ctDNA. Another preparation yielded a sperm with 10 nucleoids (Fig. 9). There is no evidence of any unusual rate of accumulation of ctDNA in (androgonidia prior to cleavage, with the result that a significant number of sperm did not appear to receive &DNA. These observations sug-

FIG. 9. Phase and fluorescence illustrating nuclei and ctDNA DAPI. x1700.

micrographs of V. carteri sperm cells, nucleoids of plastids, stained with

gest that newly formed androgonidia receive a complement of ctDNA which, although greater than that of somatic cells, ranges widely. Effect of Sexual Reproduction Somatic Cells

FIG. 8. Phase and fluorescence micrographs of completed 64-cell sperm packet of V. carteri, stained with DAPI. Although not all ctDNA nucleoids can be seen in this limited plane of focus, there appeared to be less than 100 in the ent.ire sperm packet. X1700.

Cycle on ctDNA

of

Male V. aureus spheroids bearing androgonidia in the final stages of sperm packet formation were examined to see if they showed any marked change in the characteristics of their somatic cell ctDNA. More than half the spheroids in this culture were engaged in sperm formation. No significant difference was found between the number of chloroplast nucleoids per somatic cell in male

448

DEVELOPMENTAL BIOLOGY PLASTID

DNA

CYCLE

s

----

---*

I

1

0

48

24

M

72 .

G1 NUCLEAR

cGN DNA

CYCLE

FIG. 10. Diagram of the timing of nuclear and of plastid DNA regulation during the asexual cell cycle of V. carteri. 0, 24, 48 and 72 represent hours elapsed since the completion of the previous series of cleavages. Dashed lines represent periods which were not measured. (S-M)N = represents the periods where rounds of nuclear DNA replication, alternating with mitosis, result in the formation of a new spheroid, i.e., embryogenesis.

spheroids as compared to asexual spheroids. The mean for somatic cells of asexual spheroids was 4.79 f 2.56% and that for somatic cells of male spheroids was 5.26 f 3.02%. In each type, 5-6% of the somatic cells had no visible ctDNA nucleoids. Hence, spheroids bearing androgonidia had somatic cell ctDNA nucleoids in quantities similar to those in somatic cells of asexual spheroids. DISCUSSION

The only diploid cell in the Voluor life cycle is the zygote. The data presented here suggest that nuclei of somatic cells, sperm cells and even fully developed gonidia contain the same amount of DNA, the haploid lc value. The behavior of the nucleus during the cell cycle of Voluox species such as V. aureus is relatively conventional, with increase in cell size followed by one nuclear DNA doubling, cell division, and another period of increase in cell size, in a repetitious cycle leading to a final cell number typical of a colony. The nuclear cell cycle in V. carteri, is more complex. A prolonged gonidial growth stage precedes the first doubling of nuclear DNA and cell division, and successive nuclear doublings and divisions ensue rapidly, completing colony formation in 3-4 hr time (Fig. 10). This type of cycle has also been reported for Eudorina (Lee and Kemp, 1975), Pleodorina (Kemp et al., 1979), Pandorina (Coleman, 1979b), and Chlamydomonas (Coleman, 1982); there is no evidence from these microspectrophotometric evaluations for a polyploid or polytene stage in any of these genera of the Volvocales, despite earlier conclusions to the contrary by Tautvytis (1976), based on chemical measurements on Eudorina. Biochemical analyses of DNA synthesis in Voluox carteri (Kochert, 1975; Margolis-Kazan and Blamire, 1976) have suggested that nuclear DNA synthesis

VOLUME 94, 1982

in gonidia is greatest at the time of cleavage, but occurs to some extent throughout the asexual life cycle. The apparent synthesis during gonidial expansion may represent some aspect of turnover, or asynchrony in the culture, since we find no evidence for even a doubling of nuclear DNA during this period. With respect to somatic cells, there are few biochemical data available and their interpretation is difficult. We find no evidence that either nuclear or chloroplast DNA increases in amount after colony inversion. Kemp et al. (1979), who measured nuclear DNA in somatic cells of the related genus Pleodorina (Eudorina) californica, also concluded that somatic cells maintain the lc level of nuclear DNA. Although it is possible to observe and measure quantitatively by microspectrophotometry as little as 10’ daltons of fluorochrome-stained DNA (Coleman et al., 1981), the approximate amount found in a single chloroplast genome (Gillham, 1978), both our observations on a variety of cells, organelles, and viral types, and the quantitative data presented here suggest that nucleoids contain an amount of DNA equivalent to several or many basic genomes. The Voluox carteri haploid nucleus contains about 2 X lo-l3 g of DNA. Ignoring the slight effect which differences in base composition might cause in DAPI-DNA fluorescence yield, the somatic cell plastid contains about 5% as much DNA as is found in the nucleus, a value comparable to that reported by Kochert (1975). Assuming the Voluox ctDNA genome is similar to that of Chlamydomonas, 1.2 X lo8 daltons, a single genome contains 2 X lo-l6 g of DNA and the Volvor carteri somatic cell contains 50 such molecules. These are distributed among, on the average, two or three nucleoids in a somatic cell, suggesting each nucleoid contains 16-25 genomes. Similar conclusions concerning the polyenergidity, the “polyploidy,” of chloroplast nucleoids have been drawn by Herrmann and Possingham (1981). Counting chloroplast nucleoid number may seem a simplistic way to compare presumed amounts of ctDNA, since the nucleoids themselves are so diverse in size and appearance and since we do not yet know what dictates their size and number (Kowallik and Haberkorn, 1971). Nevertheless, they appear to behave as stable entities in the absence of cell division. This is supported by the quantitative data on ctDNA-DAPI fluorescence per somatic cell at two different ages; in both nucleoid number and fluorescence value per cell there is little difference between the two somatic cell samples. Perhaps only rarely, possibly in some V. aureus sperm, is an individual nucleoid composed of as little as one chloroplast DNA genome. Mitochondria also contain DNA, although by analogy with Chlamydomonas mitochondria (Grant et al., 1978)

COLEMAN AND MAGUIRE

Volvox

their genome in Volvox rnight be presumed to be lo-fold smaller than that of the plastid DNA. Nevertheless polyenergidity occurs also in mitochondria (Williamson and Fennell, 1975) and would render the DNA visible if a “nucleoid” containing 10 genomes or more were present. In practice, identifiable mitochondrial DNA is not discernable in fixed Volvox cells, and it probably contributes little or nothing to our readings. During the initial stages of DAPI entry into living Voluox cells, 410 DAPI-DNA fluorescent particles are observed in those regions where eljectron microscopy has shown mitochondria to be, i.e., around the mouth of the plastid, and in the peripheral cytoplasm. These however soon disappear, perhaps because they lyse. For an examination of chloroplast DNA accumulation, Volvox carteri provides an unusually propitious material. The prolonged gonidial enlargement phase occurs in the absence of nuclear DNA increase, yet ultimately the cell makes greater than 2000 daughter cells, each of which requires its allotment of ctDNA. This unusual cell cycle magnifies the synthesis of ctDNA in the gonidium tremendously. Chloroplast DNA accumulation is detectable in the gonidium soon after gonidial formation and continues exponentially for approximately 3 days, at which time the gonidium finally enters the S phase of tlhe nuclear cycle. For technical reasons, it has not yet been possible to determine by microspectrofluorometry whether ctDNA synthesis continues through cleavage and inversion, but that possibility exists. During the gonidial expansion period covered by Fig. 7, the gonidia increased 50-fold in volume while the ctDNA increased 40-fold. The final quantity of ctDNA accumulated in the mature gonidium is approximately 2300 times that calculated to be in a single somatic cell (mature gonidium = 5.63 = log, 280 fluorescence units; whole somatic cell = 0.9 = log, 2.5 fluorescence units; 5% of whole somatic cell = 0.5 X 2.5 = 0.12 fluorescence units). Although these estimates are subject to considerable potential error, because the fluorescence of single somatic cells is insufficient to measure accurately at the low magnification necessary to accomodate gonidia, it would appear that the mature gonidium is almost fully stocked with the ctDNA necessary for all the daughter cells it will form. Furthermore, it contains much more ctDNA than nuclear DNA. No directly comparable biochemical data on gonidial DNA content are yet available. However, Kochert reported that younger gonidia, 6-8 hr before their first cleavage, contain about 25 times as much total DNA as do somatic cells, and th.at 6.5% of somatic cell DNA is &DNA. If nuclear DNA synthesis has not yet begun at this stage, then sufficient ctDNA is present in the gonidia to provide for nea.rly 500 daughter cells, and 8 hr remain to accomodate the two to three doublings of

Nuclear

and Plastid

DNA

Cycle

449

ctDNA necessary for the eventual production of a ca. 3500-cell spheroid (not 35,000-cell, a misprint on p. 72 of Kochert (1975)). However, as yet the CsCl gradient showing the greatest proportion of ctDNA to nuclear DNA in developing gonidia is in Kochert’s Fig. 17; the ctDNA is approximately 30% of the nuclear DNA. The basis for this discrepancy is unknown. In biochemical studies of V. carteri (Kochert, 1975; Margolis-Kazan and Blamire, 1976), chloroplast DNA was reported to be synthesized at a maximal rate during early stages of spheroid growth. These results were based on 32P0, and [2-3H]adenine incorporation. Our observations on nucleoid number and microspectrofluorometric measurement of ctDNA accumulation suggest rather that the ctDNA is synthesized in gonidia continuously throughout their enlargement. Since our measurements are based on observations of single gonidia at identifiable stages in the growth phase, we believe they avoid any possible technical problems associated with incomplete synchrony of a culture, pool sizes, and complexities of total DNA extraction (see MargolisKazan and Blamire (1976) for discussion). The relative cycles of the nuclear and the plastid DNA during asexual reproduction in V. carteri gonidial cells are diagrammed in Fig. 10. In Voluox, the occurrence of DNA synthesis in the gonidia, whether nuclear or ctDNA, appears to be entirely without influence on the DNA synthesis of adjacent somatic cells. Once formed by cleavage, the somatic cells do not alter their DNA quantity detectably. Some, in fact, appear to receive no ctDNA at the final stages of cleavage, as is true also for the chloroplasts of some Voluox sperm. These Voluox observations, along with results reported for vegetative cells of Acetabularia and Batophora, Dasycladalian algae (Woodcock and Bogorad, 1970; Coleman, 1979a) are the only examples yet known of chloroplasts apparently lacking ctDNA. Presumably there is some communication between nucleus and plastid, during vegetative and sexual reproduction, which ensures that adequate ctDNA has been accumulated before the onset of cleavage, and that monitors the number of rounds of nuclear DNA doubling and division during the rapid cleavage stage, such that most somatic cells receive an aliquot of ctDNA. This unusual organism presents in extremis a cell cycle coordination problem of organelle vs. nuclear DNA synthesis which in simpler form must be present in all photosynthetic eukaryotes. The language the cell uses for this communication and coordination system remains to be discovered. The apparently prokaryote type of cell cycle displayed by the ctDNA in gonidial growth is not without implications in the current discussion of the possible prokaryote origins of plastids. It will be important to determine whether ctDNA synthesis actually

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continues throughout the cleavage stages, something of which we are as yet uncertain. The genus Volvox has been often cited as a classical example of the evolution of oogamy, since its sperm and egg are so disparate in size, while apparently related forms of colonial green flagellates, such as Pandorina, are isogamous. Hence it is interesting to observe that Volvox sperm cells generally contain at least some ctDNA. However, this does not preclude the probability that Volvox will be found to display uniparental inheritance of chloroplast DNA. Both Chkzmydomonas (Sager and Lane, 1972) and Eudorina (Mishra and Threlkeld, 1968) are known to have uniparental inheritance of ctDNA, and in both genera, female and male gametes contain ctDNA (Sager and Lane, 1972; Coleman, unpublished). An additional mechanism is apparently responsible for the loss of ctDNA entering the zygote via the male gamete, perhaps some mechanism such as that suggested by Sager and Kitchin (1975) for Chlamydomonas. We wish to thank Christiana Geffin for her contribution to the gathering of data on Voluox nucleoids. This research was supported by NSF Grants DEB-76-82919, PCM-78-15783, and PCM-79-23054. REFERENCES CHIANG, K.-S., and SUEOKA, N. (1967). Replication of chloroplast DNA in Chlumydomonas reinhardi during vegetative cell cycle: its mode and regulation. Proc. Not. Acad. Sci. USA 57, 1506-1513. COLEMAN, A. W. (1978). Visualization of chloroplast DNA with two fluorochromes. Exp. Cell Res. 114, 95-110. COLEMAN, A. W. (1979a). Use of the fluorochrome 4’6-diamidino-2. phenylindole in genetic and developmental studies of chloroplast DNA. J. Cell Biol. 82, 299-305. COLEMAN, A. W. (1979b). Feulgen microspectrophotometric studies of Pandorina morum and other Volvocales (Chlorophyceae). J. Phy-

col. 15, 216-220. COLEMAN, A. W. (1982). The nuclear cell cycle in Chlamydomonas. J. Phycol. 18, 1922195. COLEMAN, A. W., MAGUIRE, M. J., and COLEMAN, J. R. (1981). Mithramycin- and 4’.6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles. J. Histochem. Cytochem. 36, 959-968. DARDEN, W. H. JR. (1966). Sexual differentiation in Volvor ourem, J Protozool. 13, 239-255. GILLES, R., BITTNER, C., and JAENICKE, L. (1981). Site and time of formation of the sex-inducing glycoprotein in Voluox carteri. FEBS Lett. 124, 57-61. GILLHAM, N. W. (1978). “Organelle Heredity,” pp. 602. Raven Press, New York.

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GRANT, D., SWINTON, D. C., and CHIANG, K.-S. (1978). Differential patterns of mitochondrial, chloroplastic and nuclear synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii. Planta 141,

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