The Chloroplast Nucleoid in Ochromonas Danica

J. Cell Sci. 16, 57

THE CHLOROPLAST NUCLEOID IN OCHROMONAS DANICA

II. EVIDENCE FOR AN INCREASE IN PLASTID DNA DURING GREENING

SARAH P. GIBBS, R. MAK, R. NG AND T. SLANKIS' Department of Biology, McGill University, Montreal H3C 3G1, Canada

SUMMARY During the first 24 h of light-induced chloroplast development in Ochromonas danica, the single plastid nucleoid increases 4-fold in volume. During this interval, the concentration of DNA within the nucleoid, as determined by eye and by counts of fibrils per /tm2 of nucleoid sectioned, remains constant. Thus, on morphological grounds, it appears that the amount of plastid DNA increases 4-fold during greening. To determine whether the chloroplasts of light-grown cells contain more DNA than the proplastids of dark-grown cells, exponentially growing cultures of dark- and light-grown cells were each labelled for exactly one generation with [:'H]thymidine. After fixation, the cells were embedded in Araldite, and serial i-/tm sections through entire plastids and nuclei were prepared for autoradiography. In this study, the chloroplasts of light-grown cells incorporated almost 4 times as much label into DNA as the proplastids did, whereas the nuclei of the dark- and light-grown cells were equally labelled. Another study showed that light-grown cells have slightly more total cell DNA than dark-grown cells. These 2 studies provide prima facie evidence that chloroplasts contain more DNA than proplastids and support the hypothesis that an increase in plastid DNA accompanies chloroplast development in Ochromonas.

INTRODUCTION There is now good evidence that chloroplasts contain a number of DNA molecules, all of which appear to be copies of each other. By labelling the DNA of the chloroplast of Ochromonas and observing the distribution of label among the progeny chloroplasts by autoradiography, we have shown that the chloroplast of light-grown cells contains at least 10 DNA molecules which segregate from each other at chloroplast division (Gibbs & Poole, 1973). Other investigators have looked at isolated chloroplast DNA with the electron microscope and have seen circular molecules approximately 40/tm in contour length in Euglena (Manning et al. 1971; Manning & Richards, 1972 a), spinach (Manning, Wolstenholme & Richards, 1972), corn (Manning et al. 1972) and peas (Kolodner & Tewari, 1972). Since chloroplasts are known to contain much more than 40 /tm of DNA (values between 300 and 3000 /tm are commonly reported), the chloroplasts of these species also must contain a number of DNA molecules. The evidence that these molecules are all identical comes from studies on the rate of renaturation of chloroplast DNA. Stutz (1970) and Kolodner & Tewari (1972) have shown using renaturation kinetics that the chloroplast DNA of Euglena and peas, respectively, has essentially the same kinetic complexity, or genetic 37-2 580 S. P. Gibbs, R. Mak, R. Ng and T. Slankis information, as a 40/fm DNA molecule of unique nucleotide sequence. The absence of any segregation mechanism similar to mitosis also supports the hypothesis that the DNA molecules of an individual chloroplast are all copies of each other. The fact that chloroplasts possess numerous identical DNA molecules allows the possibility that individual chloroplasts may possess widely different amounts of DNA yet still have a complete set of chloroplast genes. Variable amounts of DNA per plastid have been reported for plants as diverse as Acetabidaria (Woodcock & Bogorad, 1970) and sugar beet (Herrmann, 1970; Herrmann & Kowallik, 1970; Kowallik & Herrmann, 1972a). In this paper we have studied the effect of light-induced chloroplast development on the amount of DNA per plastid. Evidence is presented that the large increase in chloroplast volume which occurs during greening in Ochromonas danica is accompanied by a 4-fold increase in plastid DNA.

MATERIALS AND METHODS Culture conditions Cells of Ochromonas danica Pringsheim were grown as described in the preceding paper (Gibbs, Cheng & Slankis, 1974).

Determination of chloroplast and chloroplast nucleoid volume in dark-grown and greening cells Replica dark-grown cultures which were in the exponential phase of growth and contained between 08 and i-o x io6 cells/ml were placed in 3770 lux (350 ft-c.) of light in order to induce chloroplast development, and sample cultures were removed from the light and prepared for electron microscopy after o, 6, 12, 24, and 102 h. Half of each culture was fixed by a standard glutaraldehyde-osmium tetroxide method; the other half was fixed by a modified Ryter & Kellenberger method (1958). Details of the fixing, embedding, and staining procedures are given in the accompanying paper (Gibbs et al. 1974). The volume of the chloroplast and chloroplast nucleoid was determined on the cells fixed by the glutaraldehyde-osmium tetroxide method. First the per cent of the cell occupied by the chloroplast and chloroplast nucleoid was determined. For each time interval, approximately 100 random cell sections, each from a different cell, were photographed at 3500 x and micrographs printed at 28000 x. For large cell sections, several photographs were taken and a composite print assembled. The areas of the cell, chloroplast, and also the nucleus and vacuole were measured with a planimeter. The area of each chloroplast nucleoid sectioned was measured by a grid ruled on transparent plastic, each square of which equalled 001 /im! on the electron micrograph. Per cent volumes were converted to absolute volumes using the mean cell volume of living cells at each stage of greening. This was determined using cells in complete growth medium (Aaronson & Baker, 1959) with a Model ZI^ Coulter Counter calibrated with paper mulberry pollen. Estimates of the amount of DNA present per area of nucleoid sectioned were made as described in the text using the cells fixed by the Ryter and Kellenberger method.

Light-microscopic autoradiography of serial sections of light- and dark-grown plastids Cultures of dark- and light-grown cells which were in the exponential phase of growth were each labelled for exactly one generation with 10/tCi/ml [Me-3H]thymidine (New England Nuclear Corp., Boston, Mass.; sp. act. 195 Ci/mmol). The isotope was added under a dim green safelight to a dark-grown culture when it contained 1-4 x ioe cells/ml and the cells were incubated in the dark in the presence of isotope until the cell number had doubled (13-3 h). Similarly, isotope was added under sterile conditions to a light-grown culture when it contained 4-2 x io6 cells/ml and the culture was incubated in the light in the presence of isotope Increase in chloroplast DNA during greening 581 for one doubling time (98 h). After washing once in unlabelled medium, both cultures were fixed in 3 % glutaraldehyde in 005 M potassium phosphate buffer, pH 7-3, for 12 h at 4 °C, rinsed for 4 h in 8 changes of cold buffer, and postfixed in 2% osmium tetroxide in 01 M potassium phosphate buffer, pH 7-3, for 80 min at 4 °C. After blocking in agar, the cells were dehydrated in a graded ethanol series, followed by propylene oxide, and embedded in Araldite. Series of 6-7 consecutive i-/tm sections were cut on a Porter-Blum MT-2 ultramicrotome and mounted in order on glass slides by the technique described previously (Gibbs & Poole, 1973). The slides were dipped in Kodax NTB-2 nuclear emulsion, maintained at 40 °C, and allowed to dry very slowly in a darkroom held at 28 °C and 75 % relative humidity. The slides were stored over Drierite at 4 °C and developed in Kodax D-19 developer for 2 min at 20 °C after an exposure time of 14 days. The slides were stained with an aged solution of 1 % methylene blue in 1 % borax at 40 °C for 2 h. The slides were examined with a Zeiss microscope with a IOO X planapochromat oil-immersion lens. The grains over the nucleus, chloroplast, cytoplasm and vacuole were recorded for each cell section and the counts for each cell summed. In order to follow individual cells in serial i-/tm sections, it was usually necessary to draw diagrams of every section of each cell analysed. Counts of background grains were made using a Whipple disk.

Determination of total cell DNA In each experiment, 4 replicate samples of a dark-grown culture in the exponential phase of growth and 4 replicate samples of a light-grown culture in the exponential phase of growth were extracted for DNA by a modified Schneider procedure (Munro & Fleck, 1966). Under the growth conditions used, dark-grown cells remain in the exponential phase of growth up to 3 x io° cells/ml, whereas light-grown cells do not leave the exponential phase of growth before 10 x 10" cells/ml. The cell concentrations in the six light-grown cultures analysed ranged from 1-4 x io° cells/ml to 81 x io° cells/ml. The cell concentrations of all but one of the dark-grown cultures analysed were between o-8 and 3-0 x io° cells/ml. The dark-grown culture in Expt. 5 contained 4-7 x 10" cells/ml and thus was in the linear rather than the exponential phase of growth. The cells were collected by centrifugation at 12 100 g for 10 min at 4 °C and washed 3 times in 10% trichloroacetic acid for 10 min each at 4 °C. The cells were then extracted at 80 °C for 10 min each in the following series of lipid solvents: 10% potassium acetate in 95% ethanol, 95% ethanol, and 2 changes of 3:1 ethanol:chloroform. Between each extraction, the cells were collected by centrifugation at 12000 g for 10 min. The cells were then suspended in 1:1 ethanol:ether and allowed to evaporate to dryness overnight at room temperature. The residue was extracted in 5 ml of 05 N perchloric acid for 20 min at 70 °C. After centrifugation, the supernatant was analysed for deoxyribose by Burton's (1956) diphcnylamine method. Three replicas were analysed for each sample. DNA concentrations were determined from standard curves prepared from calf thymus DNA (Sigma Chemical Co., St Louis, Mo.).

RESULTS Increase in volume and total fibril content of the plastid nucleoid during light-induced chloroplast development In the preceding paper, we have shown that the plastid nucleoid in both dark- and light-grown cells of Ochromonas danica has the shape of a cord, or ring, which encircles the rim of the plastid just inside the girdle thylakoid or girdle bands (Gibbs et al. 1974). Since during light-induced chloroplast development, the small proplastid grows considerably in volume (Gibbs, 1962), it is obvious that the chloroplast nucleoid must lengthen as the perimeter of the plastid increases. Thus, unless there is a corresponding reduction in the cross-sectional diameter of the nucleoid, the total volume of the chloroplast nucleoid must increase during light-induced chloroplast development. 582 S. P. Gibbs, R. Mak, R. Ng and T. Slankis To verify this quantitatively, log-phase dark-grown cells were placed in the light to green and samples were fixed for electron microscopy at appropriate intervals. The mean volume of the chloroplast nucleoid at each stage of development was determined from random sections of glutaraldehyde and osmium tetroxide-fixed cells. The results are given in Table 1. It can be seen that during the first 6 h in the

Table 1. Changes in plastid and nucleoid volumes during light-induced chloroplast development

Chloroplast nucleoid Chloroplast volume volume

\ 1 \ 3 Hours in light volume, /im % of cell /(m3 % of cell /*m:l

0 45° 3-0 13-5 0-17 076 0* 45O 2-4 108 0-15 068 6 545 5'5 30-0 046 25 12 560 6-8 381 0-50 28 24 560 99 55-4 0-50 28 102 560 15-0 84-0 0-12 0-67 # Log-phase dark-grown cells from a different experiment.

Table 2. Changes in nucleoid dimensions during chloroplast development

Average Hours in Average length, light diameter, //.m //.m

0 O-2I 22 6 026 48 12 0-27 5° 24 0-24 60 102 009 97 light, the chloroplast nucleoid increases 3-5-fold in volume while the chloroplast itself increases 2-5-fold in volume. By 12 h in the light, the chloroplast nucleoid has increased approximately 4-fold in volume, whereas the chloroplast has increased 3-fold in volume. During the next 12 h in the light, the volume of the chloroplast nucleoid stays constant, whereas the chloroplast continues to grow (by 24 h light the chloroplast has increased 4-6-fold in volume). In this experiment, the cells stayed in the exponential phase of growth during the first 48 h in the light and entered the stationary phase shortly thereafter. It is interesting that at some time between 24 and 102 h in the light, the chloroplast nucleoid reverts in size to essentially the same volume it had in the dark-grown cells (Table 1). Table 2 gives the average cross-sectional diameter of the chloroplast nucleoid and its average length at different stages of chloroplast development. The average diameter was determined from measurements made on sections of the nucleoid which were judged to be true cross-sections. The length of the nucleoid was then calculated from the known nucleoid volume. It can be seen that during the first 12 h of chloroplast Increase in chloroplast DNA during greening 583 development, there is a marked increase in the length of the nucleoid as well as a slight increase in its cross-sectional diameter. Thereafter, the nucleoid becomes both thinner and longer. The crucial question is whether the 4-fold increase in the volume of the chloroplast nucleoid which occurs during the first 12 h in the light is accompanied by a corresponding increase in the amount of chloroplast DNA or whether the increase in nucleoid volume simply represents a loosening or spreading out of the existing DNA fibrils. Figs. 1-3 arc representative sections of the plastid nucleoids in Ryter & Kellenbcrger-fixed cells after 0, 6, and 12 h in the light, respectively. These 3 figures should be compared with fig. 3 of the preceding paper (Gibbs et al, 1974), which is a cross-section of the chloroplast nucleoid of a greening cell which has been in the light 24 h. As far as can be determined by eye, the nucleoids of the 6-, 12-, and 24-h cells have just as much DNA per unit area as does the nucleoid of the dark-grown cell. In order to quantify this observation, one of us (T. Slankis) employed a consistent, although arbitrary, method of determining the amount of DNA per unit area. A DNA fibril was defined as a strand of DNA between 2 intersections or a strand having 2 free ends. Preliminary measurements showed that the average length of a fibril so defined was approximately the same in each sample analysed. For each sample, the fibrils of 50-100 nucleoid sections were counted. In order not to count a fibril more than once, each fibril was marked on a plastic overlay as it was counted. The area of each nucleoid section was determined using a ruled grid. The results obtained confirmed our visual impression that the amount of DNA per area of nucleoid does not vary during the early stages of greening. The nucleoid of dark-grown cells contained 258 fibrils//(m2, that of 6-h-light cells contained 286 fibrils//(m2, whereas the nucleoid of 24-h-light cells had 259 fibrils//tm2. Thus it appears that during the first 24 h of light-induced chloroplast development, the amount of DNA per chloroplast increases 4-fold in parallel with the increase in the volume of the chloroplast nucleoid. Fig. 4 is a longitudinal section of the chloroplast nucleoid in a cell which had been in the light for 102 h. The DNA fibrils in this nucleoid appear more concentrated than those in the nucleoids of dark-grown and early greening cells (cf. Figs. 1-3). Counting these fibrils was difficult and probably not very reliable, but a value of 434 fibrils//^2 was obtained. This indicates that the ehloroplasts of 102-h-light stationary phase cells have roughly 40% of the DNA content of the ehloroplasts of 24-h-light log-phase cells.

Amount of label incorporated into plastid DNA in dark- and light-grown cells during one generation in [3H]thymidine If the ehloroplasts of light-grown cells do contain more DNA than the proplastids of dark-grown cells, then one would expect that light-grown cells would incorporate more [:iH]thymidinc into plastid DNA during one generation's growth in isotope than would dark-grown cells. Consequently, an exponentially growing culture of light-grown cells was labelled in the light for one generation, or 9-8 h, with [Me-3H]- thymidine. An exponentially growing culture of dark-grown cells was similarly labelled in the dark for one generation, or 13-3 h. Both cultures of cells were then 584 S. P. Gibbs, R. Mak, R. Ng and T. Slankis fixed in glutaraldehyde and osmium tetroxide, embedded in Araldite, and prepared for autoradiography. Previous deoxyribonuclease digestion studies of cells of Ochromonas labelled with [Me-3H]thymidine and prepared for autoradiography by the same methods have established that virtually all cell grains arise from labelled DNA (Gibbs & Poole, 1973). In order to measure quantitatively the relative amounts of label incorporated into proplastids and chloroplasts, it was necessary to use autoradiographs of serial sections through entire plastids. Serial i-/tm sections were cut, and 6 or 7 of these were mounted on glass slides and prepared for autoradiography. Only those cells which were sectioned at the level of the nucleolus in the middle section were used in the analysis. In such cells, the entire proplastid and the entire nucleus were always sectioned. However, in some light-grown cells, the entire chloroplast was not included in the series, so the observed grain count for the chloroplasts of light-grown cells is actually slightly lower than it should be.*

Table 3. Amount of label incorporated into different cell organelles after labelling exponentially growing cells with [3H]thymidine for one generation

Grains/total No. of Grains/total No. of Cytoplasmic Growth plastid,* plastids nucleus,f nuclei grains/section, conditions mean ± s.E. analysed mean ± S.E. analysed mean ± S.E.

Dark 27 ± 02 78 7-1 + 0-5 75 1-3 ± 009 Light 84 ± 05 102 8-i ± 06 105 13 ± 008 * The average proplastid of dark-grown cells occupied 4/1 serial sections, whereas the aiverage chloroplast of light-grown cells occupied 5-8 sections. f The average nucleus was present in 3-4 serial sections in dark-grown cells and in 36 sections in light-grown cells.

The results obtained are shown in Table 3. It can be seen that the chloroplasts of light-grown cells contain 3-1 times as much label as the proplastids of dark-grown cells. When the observed chloroplast grain count is corrected for the fact that not all series included an entire chloroplast, the chloroplasts of light-grown cells are seen to contain 3-7 times as much label as proplastids.* On the other hand, there is not a significant difference (P > 0-05) in nuclear labelling between the dark- and light- grown cells. Nor is there any difference in cytoplasmic labelling (which presumably represents mitochondrial DNA synthesis) between the dark- and light-grown cells. Background grain counts in both experiments were 0-14 grains/100/tm2 of section. The volume of an average chloroplast in cells of Ochromonas danica growing exponentially in the light is 76 /mi3 (Gibbs & Poole, 1973). Knowing the shape of the chloroplast, it is possible to calculate the area occupied by the chloroplast when cut in * In another autoradiographic experiment on log-phase light-grown cells (Gibbs & Poole, IO-73)> I0 to 12 serial i-/tm sections were employed. In that case, the entire chloroplast was virtually always sectioned in those cells which were cut at the level of the nucleolus in the middle section. The average chloroplast in that experiment occupied 69 sections rather than 58 sections. Thus, as a rough estimate, the number of grains per plastid in the light-grown cells would have been approximately 20 % higher than the observed value given in Table 3 had a longer series of sections been employed. Increase in chloroplast DNA during greening 585 6 or 7 sections. The precise value varies depending on the angle at which the chloroplast is cut, but is almost always less than 100 /tin2. Clearly, then, the number of background grains found over an entire chloroplast is insignificant. Since the average nucleus is only 18/tm3 in volume, and the average proplastid, only 12 /tm3, background labelling of these organclles is also negligible. The most direct explanation for the observation that the chloroplasts of light- grown cells incorporate almost 4 times more [3H]thymidine into DNA in one generation than do the proplastids of dark-grown cells is that chloroplasts contain 4 times as much DNA as proplastids. However, for this explanation to be valid, a number of assumptions must be met. These are discussed below. First of all, it is necessary to rule out the possibility that chloroplasts of light- grown cells are more heavily labelled because there are several rounds of replication and concomitant breakdown of chloroplast DNA in one generation in the light, but only one round in the dark. Manning & Richards (19726) have suggested that in light-grown cells of Euglena one-third of the chloroplast DNA turns over each cell generation. However, we have shown that in light-grown cells of Ochromonas there is no breakdown of labelled chloroplast DNA during 3-3 generations in unlabelled medium (Gibbs & Poolc, 3973). The opposite alternative that a significant percentage of the proplastid DNA complement is not replicated in one generation can also be ruled out, for dark-grown cells do not lose their proplastid DNA. A second factor which must be considered is whether the radioactivity present in the proplastids and chloroplasts is recorded in the photographic emulsion with the same efficiency. The chloroplast has a greater density than the proplastid which would indicate that there would be more grains lost due to self-absorption in the chloroplast than in the proplastid. However, in Araldite-embedded tissue, the self-absorption of the ft particles is primarily due to the Araldite itself (Hodson, 1969), so that the differences in density of chloroplast and proplastid have little effect on the grain counts observed. The radioactive source, the nucleoid, has the same basic shape in both dark- and light-grown cells, except that in light-grown cells, the nucleoid is longer and cut in more sections. When the nucleoid is sectioned perpendicularly, the grain counts observed will reflect the concentration of tritium in the nucleoid. When the nucleoid is sectioned tangcntially, the observed grain count will depend on the depth of the nucleoid in the section due to the self-absorption of tritium. There will thus be differences in the efficiency with which the radioactivity present in individual plastids is recorded depending on the geometry of the nucleoids within the serial sections. However, when a large number of proplastids cut in serial sections are compared with a large number of chloroplasts, the grain counts observed will be proportional to the radioactivity present. Finally, the conclusion that chloroplasts of light-grown cells contain almost 4 times as much DNA as proplastids only holds if the specific activity of the tritiated thymidine is the same in the chloroplast pool as it is in the proplastid pool. Unfortunately, we have no direct data on this. We can probably rule out the possibility that light affects the uptake of thymidine at the cell membrane since the nuclei of light- and dark- grown cells are equally labelled. However, we cannot rule out the possibility that the 586 S. P. Gibbs, R. Mak, R. Ng and T. Sla?ikis sizes of the thymidine pools within the chloroplast and proplastid may be markedly different or that the rate of uptake of exogenous thymidine at the plastid envelope may be different in light- and dark-grown cells. Nonetheless, in the absence of knowledge of plastid pool sizes, the observed greater labelling of chloroplasts of light-grown cells can be taken as prima facie evidence that chloroplasts have more DNA than proplastids.

DNA content of dark- and light-grown cells The most direct way to determine whether the chloroplasts of light-grown cells contain more DNA than do the proplastids of dark-grown cells would be to obtain preparations of unbroken chloroplasts and proplastids, free of contamination by nuclear DNA, and to determine the amount of DNA per plastid in each preparation. Unfortunately, we have not been able to obtain preparations of unbroken chloroplasts from Ochromonas. Preliminary experiments also showed that chloroplast DNA of

Table 4. DNA content of exponentially growing cells

Dark-grown cells, Light-grown cells, Ratio, Expt. no. fg*/cell + s.E. fg/cell ± S.E. Light/dark

I 232 + 3 266 ± 5 ] 15 2 243 ± 8 275 ± 4 •13 3 243 ± 9 292 ± 17 •20 4 227 ± 4 276 ± 19 •22 5 236 ± 3 320 ± 11 •36 6 207 ± 6 302 + 10 •46 Average 231 289 •25 * 1 femtogram = io->5g.

Ochromonas has the same buoyant density in CsCl as nuclear DNA. Therefore, it has not been possible to determine the comparative amounts of plastid DNA present in dark- and light-grown cells from CsCl gradients. Consequently, we looked at the total amount of DNA in cells growing exponentially in the dark compared with that in cells growing exponentially in the light in the expectation that if chloroplasts do indeed have more DNA than proplastids, light-grown cells should have slightly more DNA per cell than dark-grown cells. Table 4 shows that light-grown cells contain on average 25 % more DNA than dark-grown cells. An analysis of variance on the six experiments showed that the difference between the amount of DNA in light- and dark-grown cells is highly significant whether this difference is compared with the variability in the light/dark ratios between experiments (P < o-oi) or whether this difference is compared with the variability of the individual observations within each experiment (P < o-ooi). The results presented in the preceding sections of this paper suggest that the greater amount of DNA found in light-grown cells is due to the presence of a greater amount of plastid DNA in these cells. It could be argued, however, that since most of the Increase in chloroplast DNA during greening 587 DNA in the cell is nuclear DNA, the small difference observed in total cell DNA between dark- and light-grown cells might represent a variation in the amount of nuclear DNA per cell. For example, since the DNA extractions were made on asyn- chronous populations of exponentially growing cells, then if the period of nuclear DNA synthesis comes proportionally earlier in the cell cycle in light-grown cells than it does in dark-grown cells, light-grown cells would have significantly more nuclear DNA. However, if this happened, one would expect that the nuclei of the light-grown cells in the autoradiographic experiment would be more heavily labelled than those of the dark-grown cells. But, in fact, in that experiment, the nuclei of dark- and light-grown cells were equally labelled.

DISCUSSION In this paper we have presented a variety of evidence that the amount of DNA present in the single plastid of Ochromonas increases during the early stages of light-induced chloroplast development. The most conclusive evidence for this asser- tion is that the plastid nucleoid increases 4-fold in volume during the first 24 h in the light without there being any observable change in the concentration of DNA fibrils within the nucleoid. It is, of course, possible that there is a large loss of plastid DNA fibrils during the fixing and processing of dark-grown cells and little or no loss of DNA fibrils during the fixingo f light-grown cells, but this is unlikely. Further support for the hypothesis that the amount of plastid DNA increases during greening comes from the observation that chloroplasts of light-grown cells incorporate almost 4 times as much labelled thymidine into DNA as do dark-grown proplastids when both are exposed to isotope for one generation. Although labelling studies of this type are always subject to uncertainties due to the fact that the concentration of radioactive thymidine within the plastid pools is not known, nonetheless, the close correspondence between the electron-microscopic observations and the autoradiographic results supports the hypothesis that green chloroplasts do have more DNA than proplastids. Further support for the hypothesis is provided by the observation that light-grown cells have slightly more total cell DNA than do dark-grown cells. We (Gibbs & Poole, J973) have previously shown that the chloroplast of light-grown cells of Ochromonas contains a minimum of 10, and probably 20, separate DNA molecules, which are presumably copies of each other. Thus, the observed increase in the amount of DNA per plastid during greening probably represents a simple increase in the number of DNA molecules per plastid. In genetic terms, this would represent an increase in the ploidy of the single chloroplast nucleoid. The possibility that selective amplifi- cation of chloroplast ribosomal genes occurs during greening seems to us to be less likely, but certainly warrants further study. The previous studies on whether the amount of DNA per plastid changes during development have come to contradictory conclusions. Gyldenholm (1968) looked at the amount of DNA per plastid in proplastids and chloroplasts isolated from greening leaves of Phaseolus vulgaris and found no change during the 50-h greening interval. However, Gyldenholm observed very large amounts of DNA per plastid (1-4 x io~14 g), 588 S. P. Gibos, R. Mak, R. Ng and T. Slankis so it is probable that his plastid fractions were contaminated with considerable amounts of nuclear DNA which would have masked any variation in plastid DNA. Olszewska & Mikulska (1964) studied the incorporation of labelled thymidine into the plastids of three successive segments of the basal regions of the leaves of two monocotyledons, Clivia viiniata and Bilbergia sp. The first segment contained colour- less proplastids, the second moderately green chloroplasts, and the third fully green chloroplasts. They observed that the chloroplasts of the second and third segments were well labelled, but the proplastids were unlabelled. One interpretation of their data is that the proplastids of these plants, like the proplastid of Ochromonas, contains relatively little DNA. However, their data may only mean that the proplastids in the first leaf segment divide very rarely and thus did not replicate DNA in the 8-h labelling period employed. By far the most comprehensive studies on the variation in plastid DNA content of chloroplasts of different sizes and at different stages of development are those of Herrmann & Kowallik on Beta vulgaris. These investigators studied both euploid and trisomic plants of sugar beet grown in alternating light-dark cycles. The chloroplasts of the trisomic plants are on the average considerably larger than those of the euploid plants. However, as they studied the chloroplasts found in a series of young leaves of each type of plant, they observed a variety of chloroplast types within each plant, ranging from very small undifferentiated plastids to large mature chloroplasts. In the autoradiographic studies, Herrmann (1969, 1970) incubated isolated leaves from each type of plant in a solution containing labelled thymidine and showed that there was an approximate proportionality between the cross-sectional area of each chloroplast analysed and the number of silver grains it contained. DNase-digestion controls showed that most of the silver grains arose from labelled DNA. Thus large chloroplasts appeared to have considerably more DNA than small ones. Electron- microscope studies of the same plants made using serial sections (Herrmann & Kowallik, 1970; Kowallik & Herrmann, 1972a) showed that large chloroplasts contained many more DNA regions, or nucleoids, than did small undifferentiated plastids. In addition, biochemical analyses of the DNA content of chloroplasts isolated according to size have confirmed that the large chloroplasts of Beta vulgaris contain much more DNA than do the small plastids (R. G. Herrmann, personal commu- nication). An analogous situation occurs in rat liver mitochondria. Bahr (1971) has shown that the DNA content of mitochondria isolated by size is closely proportional to their dry weight. In the algae, a large variation in the DNA content of individual chloroplasts has been reported for Acetabularia mediterranea and its close relative, Polyphysa cliftoni (Woodcock & Bogorad, 1970). Although Woodcock & Bogorad report that a large percentage of the chloroplasts of these algae contain no DNA at all, we believe that most, if not all, of the chloroplasts reported to contain no DNA did indeed contain small amounts of DNA which were not detected by the methods used. However, whichever interpretation is correct, it seems indisputable that individual chloroplasts of Acetabularia and Polyphysa contain widely differing amounts of DNA. There is also some evidence in the literature that mature chloroplasts of vascular Increase in chloroplast DNA during greening 589 plants may lose some of their DNA as they age. Yokomura (1967) has reported that the chloroplasts of old leaves of a variety of plants appear under the electron microscope to have fewer DNA areas than do the chloroplasts of young leaves. Kowallik & Herrmann (19726) have observed that the chromoplasts of Narcissus appear to contain fewer DNA regions than do the chloroplasts from which they differentiated. Thus it appears that in ageing plant tissues either chloroplast DNA breaks down or chloroplasts divide one or several times without replicating their DNA. That the latter can happen experimentally has been demonstrated by Boasson & Laetsch (1969) who showed that the chloroplasts of tobacco leaf disks incubated in the presence of 5-fluorodeoxyuridine continued to divide several times in the absence of DNA synthesis. The resulting chloroplasts, although normal in structure, were much smaller than the parent chloroplasts and presumably contained less DNA. It seems likely that the apparent reduction of chloroplast DNA which occurs in stationary-phase green cells of Ochromonas is brought about by a chloroplast division occurring without a concomitant round of DNA synthesis.

We wish to thank Mrs Lily Chu for her skilful assistance in the autoradiographic study. This research was supported by the National Research Council of Canada (Grant no. A-2921) and la Commission de la recherche scientifique du Quebec.

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Figs. 1-4. Longitudinal sections of the chloroplast nucleoid in Ochromonas cells fixed by the Ryter & Kellenberger method, showing the comparative concentration of DNA fibrils at different times during greening, gb, girdle band, x 78400. Fig. 1. Log-phase dark-grown cell, 0-9 x io6 cells/ml. Fig. 2. Log-phase greening cell, 6 h light, rox 10" cells/ml. Many of the DNA fibrils appear to come into contact with the ribosomes at the nucleoid's inner border (arrows). Fig. 3. Log-phase greening cell, 12 h light, 1-2 x ioe cells/ml. In this section several clusters of ribosomes are found within the nucleoid in close association with DNA fibrils. Fig. 4. Stationary-phase green cell, 102 hlight, 15-6 x io6 cells/ml. In this figure, the chloroplast DNA fibrils appear to be less distinct and more concentrated than in the preceding figures. Increase in chloroplast DNA during greening