Current Genetics

Current Genetics 1,127-131 (1980)

©

by Springer-Verlag 1980

Chloroplast Gene Inheritance Studied by Somatic Fusion in Chlamydomonas reinhardtii Ren6 F. Matagne and Marie-Pattie Hermesse

Laboratory of MolecularGenetics,Department of Botany, Universityof Liege,Sart Tilman, B-4020 Li[ge, Belgium

Summary. Somatic fusion between strains of Chlarnydomonas containing complementing cell-wall and auxotrophlc mutations, having the same mating-type (mr) and bearing chloroplast markers, have been performed to study the mode of chloroplast gene inheritance in the fusion products. About one third of the fusion products (rnt÷/mt+ or m t - / m t - ) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other (uniparental fusion products) exclusively. Incubation of the fusion products in the dark for 48 hours, immediately after the fusion, decreases the frequency of biparental fusion products. The results indicate that the general process of elimination of chloroplast alleles is independent of the presence of both mt ÷ and m t - alleles in the cell. In contrast, directional elimination (i.e. preferential elimination of paternal chloroplast alleles) does appear to depend upon heterozygosity at the mt locus. These results are discussed in relation to the models which have been proposed to explain the maternal inheritance of chloroplast genes in Chlamydomonas. Key words: Somatic.

Chlamydomonas

-

Chloroplast - Fusion -

Introduction

The isogamous green alga Chlamydomonas reinhardtii has proven to be a model organism for the study of chloroplast heredity. Since the original study by Sager (1954)

Offprint requests to: R. F. Matagneat the above address.

of uniparentally-inherited mutants resistant to streptomycin, many mutants exhibiting a similar non-mendelian pattern of inheritance have been isolated (for a recent review, see Gillham, 1978). These non-mendelian genes, thought to reside in the chloroplast DNA, are inherited almost exclusively from the mating-type plus (rot+) or maternal parent: more than 90% of the zygotes produce haploid offspring all of the maternal genotype (maternal zygotes). A few exceptional zygotes transmit to their progeny chloroplast alleles from both parents (biparental zygotes) and very rarely only the alleles of the mating-type minus (rot-) parent (paternal zygotes). Some of the meiotic products derived from biparental zygotes are still heteroplasmic and continue to segregate the chloroplast alleles during the subsequent mitoses until nearly all progeny are homoplasmic by the completion of 10-20 generations. The chloroplast genes are transmitted in a distinctly different manner in stable diploids (Gillham, 1963; VanWinkle-Swift, 1978): 50-90% of the vegetative zygotes are biparental for chloroplast genes and segregate both maternal and paternal alleles to their diploid progeny. The rest of the vegetative zygotes are uniparental, uniparental maternal inheritance being generally more common than uniparental paternal. VanWinkle-Swift (1978) observed that when the first mitotic division of the vegetative zygote was delayed (for example by selection of the zygotes in darkness), the frequency of biparental vegetative zygotes decreased and the pattern of chloroplast gene transmission shifted towards uniparental maternal inheritance. Two distinctive models have been proposed to explain the inheritance of chloroplast genes in Chlamydomonas. Both have been extensively discussed in a recent review of the problem (Gillham, 1978). Sager and Ramanis (1973) and Sager (1977) proposed a model according to which the chloroplast genes are located in

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R. F. Matagne and M.-P. Hermesse: Chloroplast Inheritance Following Somatic Cell Fusion

two circular copies of the genome. The mt+ gametes contain an inactive modification enzyme (M) whereas the m t - gametes contain an inactive restriction enzyme (R). Activation of M and R is regulated by substances (G1 and G2) produced by m t + during gametogenesis. During the first hours of zygote formation, before chloroplast fusion, the mt+ chloroplast DNA is modified as G 1 enters the m t + chloroplast and activates M, whereas the m t - DNA is degraded through activation of R by G2. Biparental inheritance results from occasional failure to synthesize or activate the restriction enzyme. Gillham et al. (1974) proposed a multicopy model based on the principle of competition between incompatible plasmids. The model assumes that the chloroplast genes are localized in the numerous identical DNA copies (~- 40 in gametes, 80 in vegetative cells) known to be present in the chloroplast (Gillham, 1978). The zygote, contains a limited and fixed number of membrane attachment sites required f o r replication and transmission of chloroplast genomes. The sites are occupied preferentially by maternal chloroplast genomes. Genomes which are not attached are not replicated and are then lost either by dilution or destruction by a nuclease. Occasional detachment of maternal genomes and their replacement by paternal genomes accounts for instances of biparental inheritance. From experiments with ultra-violet irradiation of mt+ or r o t - gametes, Adams (1978) recently proposed a random choice model: the zygote contains about 27 copies of maternal genetic units (with a 95% confidence interval of 11 to 63 copies) and only 2 copies of the paternal genetic units. Sometime prior to meiosis, two genetic units are randomly selected from the population, the mechanism responsible for the selection remaining to be identified. Because of the excess of maternal genomes, there is a high probability that both of the randomly chosen genetic units will be of maternal origin. More recently, Birky (1978 and in preparation) has proposed that repeated rounds of genome pairing and gene conversion in the zygote could lead to a random drift in gene frequencies with the eventual fixation of one parental chloroplast allele and concommitant loss of the other. In the absence of differences in the initial frequencies of opposite parental alleles or directional forces (such as modification), each class of uniparental zygote would be equally frequent. None of the various models is completely satisfactory in explaining numerous genetical and biochemical data now available (for discussion of the strengths and weakness of each model, see Birky, 1978, and Gillham, 1978). We recently demonstrated somatic fusions between mutant cells deprived of their cell-walls (= stable protoplasts) and the formation o f stable diptoids in C h l a m y d o m o n a s (Matagne et al., 1979). This technique has allowed us to isolate fusion products from cells of the

Table 1. Strains used in the fusion and cross experiments. CWl5 and CWd are two cell wall mutations known to complement and restore the formation of a normal wall in diploids (Matagne et al. 1979); arg-7-2 and arg-7-7 are very closely linked mutations, leading to the inactivation of argininosuccinate lyase: arg-7 on one hand, arg-7-2 and arg-7-7 on the other hand, belong to two different interallelic complementation groups (Matagne, 1978); sr and spr are chloroplast genes for resistance to streptomycin and spectinomycin, respectively Reference number

Markersand mating-type

Antibiotic for which the strain is resistant (chloroplast genes)

303 323 326 327 328 329 330 331 350 351

cWls arg-7 m t ÷ CWt5 arg-7 r n t CW15 arg-7-2 sr mt÷ CWd arg-7-2 sr r o t CWd arg-7-2 sprmt ÷ CWd arg-7-2 spr rntCW15 arg-7-7 sr rnt÷ CW15 arg-7-7 sr r n t CWd arg-7 sprmt + CWd arg-7 spr r n t -

no no streptomycin streptomycin spectinomycin spectinomycin streptomycin streptomycin spectinomycin specfinomycin

same mating-type, thus providing new insights into the control of chloroplast heredity in C h l a m y d o m o n a s . We describe here the analysis of chloroplast gene transmission in products obtained by somatic fusion between cells of the same mating-type, in comparison to the transmission observed in vegetative zygotes obtained by crossing m t ÷ and m t - gametes.

Materials and Methods Fusions were made between cells of the same mating-type (rnt+ or rnt-) deprived of cell-waU, auxotrophic for arginine, and bearing chloroplast markers confering resistance to streptomycin or spectinomycin (Table 1). The non-Mendelian streptomycin and spectinomycin resistant mutants were kindly supplied by Dr. J. Girard (Institut de Biologie physico-chimique, Fondation E. de Rothschild, Paris, France) and were used in crosses to isolate the triple mutant strains presented in Table 1. For fusion experiments, the cells were grown on agar solidified mineral medium lacking NH4C1 and enriched with yeast extract (M - N + YE) (Loppes et al. 1972) which provides enough arginine to allow growth of the auxotrophs. The somatic cell fusions were accomplished according to the procedure previously described (Matagne et al., 1979). The fused cells were plated on agar solidified minimal (M) medium (Loppes et al., 1972) and the plates were incubated in the light (8,000 lux, 25° C) for 8-10 days until prototrophic colonies had developed. Vegetative zygotes were obtained by crossing complementing arg- gametes of opposite mating-type and plating the mating suspension on M medium. The plates were incubated under the same light and temperature conditions as in the somatic cell fusion experiments.

R. F. Matagne and M.-P. Hermesse: Chloroplast Inheritance Following Somatic Cell Fusionz Table 2. Patterns of chloroplast gene inheritance in vegetative zygotes and fusion products. In each cross or fusion experiment, 50 colonies were analysed for the presence of the antibiotic resistance and sensitivity markers Chloroplast Chloroplast inheritance marker of pattern (frequencies) each parent Crosses 326 m t ÷ 328 r a t + 303 r a t ÷ 303 r a t +

x 323 r a t x 323 m t x 327 r n t x 329 r a t -

sr x ss spr x sps ss x sr sps x spr

Fusions 326 m t + 328 m t ÷ 327 m t 329 r a t -

x 303 m t ÷ x 303 m t ÷ x 323 m r x 323 r o t -

sr x ss spr x sps s r x ss spr x sps

BP

UPm

UPp

0.92 0.72 0.80 0.80

0.06 0.28 0.06 0.16

0.02 0.00 0.14 0.04

BP

UP1

UP2

0.30 0.28 0.36 0.36

0.36 0.32 0.42 0.18

0.34 0.40 0.22 0.46

BP, biparental; UPm, uniparental maternal; UPp, uniparental paternal; UP 1 uniparental parent 1; UP2, uniparental parent 2. sr and ss, streptomycin resistance and sensitivity; spr and sps, spectinomycin resistance and sensitivity

Abbreviations:

Fusion products were analyzed for the presence of a cellwall with the light microscope (obj. 100 X). The cell volumes of the fusion products and of the diploid cells arising from vegetative zygotes were compared to wild-type haploid cells by measuring them in a Coulter electronic counter model ZF (orifice 100 #m) (Loppes et al., 1972). For DNA quantification, the cells were grown in 300 ml liquid M medium and collected during exponential growth. The DNA content was estimated by the diphenylamine reaction according to a modification of Burton's method (Giles and Myers, 1965). In order to determine the chloroplast genotypes of diploid progeny from somatic cell fusions and from vegetative zygotes, the colonies were isolated on M medium then replica plated to M medium containing the appropriate antibiotic (streptomycin: 500 mg/1; spectinomycin: 100 mg/1). To distinguish biparental fusion products or vegetative zygotes (sr/ss or spr/sps segregating both resistant and sensitive mitotic progeny) from those uniparental for the resistant allele (st/st or spr/spr segregating only resistant cells), each colony was suspended in water, diluted and plated on M medium. A sample of the resultant progeny clones was then tested for chloroplast genotypes to allow detection of the antibiotic sensitive alMe.

Results Four sexual crosses and four fusion experiments, two with m t + strains and two with m t - strains were performed (Table 2). In control experiments, (a) each strain was treated separately with the fusion solution and (b) the mixtures of two strains were treated with water only

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(which does not promote somatic cell fusion). After 4 - 5 days, diploid colonies obtained by sexual crosses were observed on the plates whereas colonies issued from the fusion products did not become visible until 8 - t 0 days after plating. No prototrophic colonies were produced in the control experiments. In each fusion experiment, 6 colonies were sampled at random for analysis of mating-type, celt volume, and the presence o f a cell-wall: all colonies were composed entirely of cells with a normal wall (formed because of comptementation between CWls and CWa markers), a mating-type identical to that of the parental strains and a cell volume ( 5 5 - 6 0 / a m 3) similar to that of diploid cells obtained by sexual mating (Matagne et al., 1979) and clearly different from the volume of haploid cells (30 ~mg). Moreover, one clone from each fusion was subcultured and the DNA content/cell was estimated: values ranging between 1.8 and 2.2 times the haploid DNA content/cell were found (data not shown). These results are in agreement with our previous findings that somatic cell fusion products are very similar if not identical (except for the mating-type) to the diploids obtained by sexual mating. It thus seems likely that fusion products most frequently result from the fusion of only two haploid cells. Moreover, electron microscopy of numerous fusion products (R. F. Matagne, unpublished) indicates that, as for diploids obtained by sexual crosses, the fused cells apparently contain only one chloroplast. Fusion products and vegetative zygotes were analyzed for their pattern of transmission o f chloroplast genes. The results (Table 2) obtained for the vegetative zygotes are in very good agreement with those obtained by Gillham (1963) and VanWinkle-Swift (1978): biparental (BP) vegetative zygotes containing the chloroplast alleles of both parents and segregating these alleles in their progeny account for more than 70% of the vegetative zygote population. The rest of the zygotes exhibited uniparental (UP) gene transmission, with maternal inheritance usually more common than paternal. The clones obtained by fusion between cells of the same mating-type can also be separated into three classes (Table 2): the UP resistant clones similar to one parent, the UP sensitive clones similar to the other parent, and the BP clones segregating resistant and sensitive alleles. Among the progeny of the BP fusion products, some cells were still BP and could further segregate the parental alleles during subsequent divisions. These results indicate that the fusion products can be either BP or UP and that the chloroplast genes segregate through mitoses while the nuclear genes do not, as has been observed for vegetative zygotes. However, in contrast to vegetative zygotes, the frequencies of the two classes of UP fusion products are approximately equal regardless of the matingtype of the strains used.

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R.F. Matagne and M.-P. Hermesse: Chloroplast Inheritance Following Somatic Ceil Fusion

Table 3. Patterns of chlorplast gene inheritance in vegetative zygotes and fusion products after dark incubation. Immediately after plating the zygotes or the fusion products, the plates were incubated in the dark for 48 h, then in the light (8,000 lux) until the isolation of colonies Chloroplast marker of each parent Cross 330 mt+ x 3 5 1 m t -

sr sps x ss spr

Fusion 331 r o t - x 351 r o t -

sr sps x ss spr

Chloroplastinheritance pattern (frequencies)

BP

UPm

UPp

0.25

0.70

0.05

BP

UP1

UP2

0.17

0.45

0.38

Abbreviations as in Table 2 Finally the frequency of BP inheritance is lower for somatic cell fusions (28-36%) than for vegetative zygote populations from sexual crosses (> 70%). This could be related to the recent experiments of VanWinkleSwift (1978) demonstrating that a delay in the first mitotic division of the vegetative zygote (induced for example by incubation in the dark), leads to a decrease in the frequency of BP zygotes and a shift in the chloroplast gene transmission pattern toward UP maternal inheritance. In our experiments, we always observed that the formation of colonies from the fusion products was delayed in comparison to that of diploid colonies from vegetative zygotes: this could perhaps be due to a delay of the first mitotic division of the fusion products. In fact, we found that, as for the vegetative zygotes, the incubation of the plates in the dark for 48 h immediately after the fusion process, followed by normal incubation in the light, resulted in a decrease in the frequency of the BP products with a concomittant increase in the frequency of uniparental clones (Table 3).

Discussion Our results and conclusions can be summarized as follows. First, uniparental inheritance, i.e. the elimination of one parental allele and the exclusive recovery of the other, is observed in somatic cell fusion products, homozygous for the m t + or m r - allele. Therefore, uniparental inheritance p e r se does not require the presence of opposite m t alleles. Second, uniparental inheritance is bidirectional in somatic cell fusion products but is predominantly unidirectional in sexual zygospores (meiotic) and to a lesser extent in vegetative zygotes. Thus the s e l e c t i v e elimination of a specific chloroplast allele does appear to depend upon heterozygosity at the m t locus.

Analysis of products of somatic cell fusion of m t + and m t - cells (in progress) should clarify whether heterozygosity at the m t locus is not only required but also sufficient for the preferential induction of uniparental maternal inheritance, or whether differential gene regulation during gamete formation plays an equally important role. Somatic cell fusions of nitrogen-starved (i.e. gametic) cells with normal vegetative cells of the samemating-type (if such a modification of procedures still allows effective fusion) may also help to define the role of nutrient depletion in directing the uniparental mechanism. The models for uniparental inheritance proposed by Sager (1977), by Gillham et el. (1974) and by Adams (1978) have in common an assumption that the uniparental mechanism involves molecular/cellular events induced during gametogenesis and/or early zygote development. The model of Birky, (1978; Birky and Thrailkill, 1979), invoking random molecular interactions leading to genetic drift and eventual uniparental inheritance, is not dependent upon such gametogenesis-specific events, nor upon differential behavior of maternal and paternal chloroplast genomes. For this very reason, the models of Sager (1977), Gilham et al. (1974), and Adams (1978) provide more specific explanations for directed uniparental inheritance (that is, uniparental maternal inheritance) as observed following sexual reproduction, while the random drift model of Birky (1978; Birky and Thrailkill, 1979) provides the simplest explanation for the bidirectional unipar ental inheritance we have ob served for somatic cell fusion products. Application of the Sager (1977) model to our data would require major changes in the model. In particular, the restriction enzyme and its activator would have to be present in both m t + and r o t - cells to explain the uniparental inheritance we observe in (+/+) and ( - / - ) fusion products. The directionality in uniparental inheritance common to sexual zygotes but missing from somatic cell fusion products could be explained ff the modification enzyme and its activator were carried by opposite mating-types, an assumption which disagrees with the most recent description of the modificationrestriction model (Sager, 1977). Moreover, even if such changes in the model are invoked, the use of a restriction enzyme to explain our data on somatic cell fusion products requires that the enzyme show limited activity since efficient restriction in the absence of modification should eliminate all chloroplast DNA and would be expected to be lethal. The Gillham et al. (1974) model suggests competition between chloroplast genomes for a limited number of membrane attachment sites as an explanation for chloroplast gene inheritance patterns, and assumes a preferential attachment of maternal chlorplast genomes. To explain the bidirectional uniparental inheritance patterns we have observed, detachment of genomes must be equal-

R. F. Matagne and M.-P. Hermesse: Chloroplast Inheritance Following Somatic Cell Fusion ly common in vegetative cells of both mating-types and maternal or paternal chloroplast genomes must be equally capable of reattachment for their preservation, replication, and transmission to the diploid progeny. The Adams (1978) random choice model explains uniparental inheritance by the random selection of two "genetic units" (e.g. chloroplast genomes) from a population of genomes heavily biased in favor to those derived from the mt+ maternal parent. To explain the bidirectional uniparental inheritance we observe in somatic cell fusion products requires that the two parent cells (which in this case are of the same mating-type) contribute equal numbers of chloroplast genomes, an assumption compatible with the Adams model. However, none of the models discussed thus far provide an adequate explanation for the increased frequency of uniparental inheritance observed following delayed division of somatic cell fusion products, vegetative zygotes (VanWirdde-Swift, 1978), or meiotic zygospores (Sears et al., 1977). In contrast, the random drift model proposed by Birky (1978), assumes that fixation of one allele and loss of the corresponding allele of opposite parental origin requires the repeated occurrence of stochastic processes such as genome pairing and gene conversion, random genome replication, or random degradation. The probability of fixation or loss of an allele increases as the number of repetitions of such events increases. Delayed division may provide an increased opportunityfor genetic drift by prolonging the period during which such stochastic events can occur prior to the initiation of segregation at the time of the first post-mating division (Birky, 1978 and in preparation; Birky and Thrailkill, 1979). In the absence of non-random directive forces, fixation or elimination of an allele within a population of chloroplast genomes should occur as often for one parental allele as for the other. Thus according to the Birky model, we would predict approximately equal numbers of uniparental-resistant and uniparental-sensitive somatic cell fusion products, as well as an increase in both classes following delayed division. Our observations fit this prediction.

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Birky (1978) notes that genetic drift cannot, by itself, explain cases of exclusively maternal uniparental inheritance common to Chlamydomonas sexual zygotes. In this case, non-random forces must act to increase the probability of genetic drift in a specific direction. The models of Sager (1977), Gillham et al. (1974), and Adams (1978) offer interesting candidates for nonrandom forces which could provide direction for the uniparental mechanism and which, according to the work reported here, are likely to involve interactions between opposite mating-type alleles and/or differential gene expression associated with the onset of sexual reproduction. Acknowledgements. We are deeply grateful to Drs Nicholas Gillham and Karen VanWinkle-Swift for their constructive criticism of the manuscript and their intellectual stimulation. References Adams, G. M. W.: Plasmid 1,522-535 (1978) Birky, C. W. Jr.: Ann. Rev. Genet. 12,471-512 (1978) Birky, C. W. Jr., ThraJlkill, K. M.: Genetics 91,111 (abstract) (1979) Giles, K. M., Myers, A.: Nature 206, 93 (1965) Gillham, N. W.: Nature 200,294 (1963) Gillham, N. W.: Organelle Heredity, pp. 1-602, New York: Raven Press 1978 Gillham, N. W., Boynton, J. E., Lee, R. W.: Genetics 78,439457 (1974) Loppes, R., Matagne, R. F., Strijkert, P. J.: Heredity 28, 239251 (1972) Matagne, R. F.: Mol. Gen. Genet. 160, 95-99 (1978) Matagne, R. F., Deltour, R., Ledoux, L.: Nature 278, 344346 (1979) Sager, R.: Proc. Nat. Acad. Sci. USA. 40, 356-362 (1954) Sager, R.: Adv. Genet. 19,287-340 (1977) Sager, R., Ramanis, Z.: Theor. Appl. Genet. 43, 101-108 (1973) Sears, B. B., Boynton, J. E., Gillham, N. W.: Genetics 86,156157 (abstract) (1977) VanWinkle-Swift, K. P.: Nature 275,749-751 (1978) Communicated by K. VanWinkle-Swift Received October 3, 1979