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J. Cell Sa. 70, 1-15 (1984) Printed in Great Britain © The Company of Biologists Limited 1984 STOCHASTIC PARTITIONING OF CHLOROPLASTS AT CELL DIVISION IN THE ALGA OLISTHODISCUS, AND COMPENSATING CONTROL OF CHLOROPLAST REPLICATION ANNETTE S. HENNIS* AND C. WILLIAM BIRKY, JR Department of Genetics, The Ohio State University, Columbus, Ohio 432 JO, U.SA. SUMMARY We asked how chloroplasts in a unicellular marine alga are replicated and partitioned at cell division so that each daughter cell will receive the appropriate number of copies. The data were obtained simply by counting chloroplasts in pairs of daughter cells immediately after cell division. The results show that chloroplast partitioning is not always equal; however, it is equal much more often than predicted by the binomial distribution of chloroplast numbers that would be expected if partitioning were strictly random. The parental chloroplasts were partitioned equally in approximately 76 % of the divisions, while in the remaining 24 % the deviations from equality were very small. To maintain a reasonable range of chloroplast numbers in the face of unequal partition- ing, there must be some form of compensating control of chloroplast replication. Our data suggest that daughter cells that receive very large numbers of chloroplasts go directly to the next division without replicating their chloroplasts, while cells with very small numbers of chloroplasts go through two rounds of chloroplast replication before dividing. INTRODUCTION When a cell divides it is essential that both daughter cells receive a complete set of genetic information, including at least one copy of each gene and chromosome. For chromosomes in the nucleus this is ensured by having each chromosome replicated exactly once and one copy delivered to each daughter cell by the mitotic or meiotic apparatus. The situation is less clear for cytoplasmic organelles that carry genes, i.e. mitochondria and chloroplasts (reviewed by Birky, 1982; Butterfass, 1979; Dyer, 1976; Heitz, 1961; Wilson, 1931). In some organisms, each cell has a single large chloroplast or mitochondrion, which is divided approximately equally in two at cytokinesis. In some animal cells with many mitochondria, those organelles are aligned along the mitotic spindle and divided more or less equally by the cleavage furrow. In other cells with many mitochondria or chloroplasts, the organelles seem to be arranged randomly in the cytoplasm and may be partitioned randomly between the two daughter cells. Certainly there is an element of randomness in the partitioning with respect to genotype of the organelles, which leads to progressive sorting out of organelle genes to produce lineages of homoplasmic cells (vegatative segregation, reviewed by Birky, 1978; Gillham, 1978; Kirk & Tilney-Bassett, 1978). •Present address: 11872 Valley View Road, Sagamore Hills, Ohio 44067, U.S.A. 2 A. S. Hennis and C. W. Birky, Jr This genetic randomness in partitioning, coupled with the lack of any visible association of organelles with the mitotic apparatus or other device for controlling partitioning, leads to the possibility that partitioning in some organisms may be numerically strictly random. According to this hypothesis, the number of chloroplasts or mitochondria that enter a daughter cell would fit the binomial distribution. The other extreme model would be numerically uniform partitioning, in which each daughter cell would always receive a fixed proportion of the parental organelles (usually half). The only extensive data relevant to this question come from studies of the partitioning of mitochondria during spermatogenesis in scorpions (Hood, Watson, Deason&Benton, 1972; Wilson, 1916, 1931), and of chloroplasts during the divisions producing guard cells in Trifolium (Butterfass, 1969). In these cases, partitioning is intermediate between the two extreme models, being equal much more often than predicted by the binomial distribution but with some variation. However, less exten- sive data on plastids in meristem cell divisions of Epilobium show no deviation from random partitioning (Anton-Lamprecht, 1967). We have studied this problem in the unicellular alga Olisthodiscus luteus. This wall- less marine alga contains many small discrete chloroplasts lying around the periphery of the cell, which are easily counted (Cattolico, 1978; Leadbeater, 1969; Liittke, 1980). Cell division can be easily synchronized in this alga; the chloroplasts replicate during interphase, well before the cells divide (Cattolico, Boothroyd & Gibbs, 1976). By counting chloroplasts in the two halves of dividing cells, or in pairs of just-divided cells, one can determine the number of chloroplasts in each daughter; the sum of those numbers is the number in the parent cell after chloroplast replication. Our data show that chloroplast partitioning in Olisthodiscus, like that of mitochondria during sper- matogenesis and chloroplasts in guard cell divisions, is not strictly uniform but is equal much more often than predicted by the random partitioning model. Because chloroplast partitioning is not always equal, every cell division tends to increase the variance of chloroplast numbers in the population of cells. Unless there is a compensating control of organelle replication, this increasing variance will eventu- ally result in cells that have too few or too many organelles for survival, and possibly cells with no organelles at all. We find that Olisthodiscus cultures do not accumulate aplastidic cells and, instead, maintain a constant variance in chloroplast numbers. We propose three models of compensating replication control; our data favour the model in which cells with large numbers of chloroplasts skip a round of chloroplast replica- tion, while cells with very small numbers go through two rounds of chloroplast replication before the next cell division. MATERIALS AND METHODS Olisthodiscus luteus Carter was stock LB2005 from the Texas Culture Collection of Algae. Growth medium O-3 was prepared as described by Mclntosh & Cattolico (1978). Cells were grown in 1-litre low-form culture flasks containing approximately 500 ml of medium or 250-ml Erlenmeyer flasks with approximately 50 ml of medium. The cultures were maintained at 16-20°C on a 12-h light/12-h dark cycle. Cultures were illuminated from above with General Electric cool white fluorescent lights to give an intensity of 400ft-candles (1 ft-candle = 10'76391x). Chloroplast partitioning at cytokinesis Fig. 1. A dividing cell of Olisthodiscus flattened under a coverslip to permit counting of chloroplasts. Cell counts were done to follow the growth of synchronized cell cultures. Approximately 500 fi\ of tincture of iodine was added per ml of sample. Each sample was counted three times in a haemocytometer chamber and the counts were then averaged. At low cell densities, cells were concentrated by centrifugation before counting. More than 200 cells were counted for each sample. To count chloroplasts in cells, approximately 50 jul of the culture was placed on a washed glass slide and covered with a 22mm2 coverslip. The droplet begins to desiccate almost immediately and the cells flatten into a single plane of focus, allowing the chloroplasts to be counted easily. The optimum time for counting is just before the cells burst, when all the chloroplasts are easily distin- guishable (Fig. 1). The counts were made at X400 magnification with a Zeiss phase-contrast microscope. For dividing cells, the numbers of chloroplasts on each side of the incipient cleavage furrow were recorded separately as the numbers of chloroplasts in the daughter cells. To obtain chloroplast counts in just-divided cells, as opposed to cells in the process of division, single cells were isolated in O-3 medium in glass depression slides just before the onset of cell division in a synchronized culture. The depression slides were placed in moist chambers made from Petri dishes lined with wet towelling. The depressions were periodically examined in a stereoscopic microscope at X 60 magnification for the presence of two cells, signalling that division was completed. The two daughter cells were placed on a slide and the chloroplasts were counted as described above. For statistical analysis, we asked if the numbers of chloroplasts in pairs of daughter cells differed significantly from those predicted by the binomial distribution. Let n be the number of chloroplasts in the parent cell and x and n —x the numbers in the daughter cells. In no case did we have a sufficiently large number of parent cells with the same value of n to do a Chi-square test, so it was necessary to pool all parents. The data were transformed using z, the cumulative distribution function of a standardized random variable. If % is a binomial function of «,l/2 then 2 = (2x—n)/V~« is approximately a normal function of 0,1. If the normal distribution is divided into k classes of equal size, then the expectation is that the z values will be equally distributed among the k classes. We calculated z for each observed x,n and determined the number of these transformed observations falling into each of the k classes. This was compared to the expected number, \/k, using the Chi- square test with k - 1 degrees of freedom. RESULTS Parameters of synchronous and asynchronous cell replication Cell samples were taken every 12 h from an Olisthodiscus culture maintained on a constant light regime to determine the growth characteristics and maximum cell A. S. Hennis and C. W. Birky, Jr • ,_—- • • 64- 32- 16- / o i - 4- / / 2- /% 1- • / / 0-5- 1 1 0 5 10 15 Time (days) Fig. 2. Growth of an Olisthodiscus culture in 0-3 medium under continuous light. densities. The culture was initiated by inoculating a sample of a stationary-phase stock culture in fresh medium to give approximately 2-5XlO3 cells/ml. The culture doubled in cell number every 24-3 h during the exponential growth phase in 0-3 medium (Fig. 2). The maximum cell density seen was 7xlO4 cells/ml, at which point the stationary phase began. These values for cultures maintained under a constant light regime parallel very closely those seen in previous studies (Cattolico et al.

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