Mutations in four chloroplast loci of Chlamydomonas reinhadtii affecting the photosystem I reaction centers

Summary In this work seven chloroplast mutations conferring a deficiency in photosystem I reaction centers have been mapped at four chloroplast loci in Chlamydomonas reinhardtii. Recombination frequencies were estimated from diploid progeny of vegetative zygotes. These four loci were scattered throughout the chloroplast genome. The three mutations at locus I were found to be tightly linked to a mutation in the rbcL gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (Dron et al. 1983). As the psaA2 gene coding for one apoprotein of the chlorophyll-complex CPI, identified by its homology with the corresponding maize gene (Fish et al. 1985), has been found close to the rbcL gene (Dron et al. 1982), the psaA2 gene could be at locus 1.     

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.     

Characterization of a cDNA encoding the 20-kDa photosystem I light-harvesting polypeptide of Chlamydomonas reinhardtii

A cDNA encoding the precursor to a major 20-kDa thylakoid polypeptide of Chlamydomonas reinhardtii (P22), previously localized to the photosystem I light-harvesting complex (LHCI), was characterized. N-terminal sequencing of P22 identified the precursor cleavage site. Genomic Southern blots and polymerase chain reaction analyses show that the gene for P22 (Lhca1^*1) is single-copy and contains at least one intron. Northernblot analyses show that Lhca1^*1 mRNA is highly regulated in light-dark synchronized cells. The primary sequence and predicted topology of P22 has features characteristic of light-harvesting chlorophyll a/b-binding proteins from higher plants. Sequence comparisons indicate that P22 has significantly greater identity with the Type-I LHCI protein of tomato, compared to other LHC proteins. This result suggests that the divergence of LHCI proteins into the classes found in higher plants may have occurred early in evolution, prior to the separation of green algae and land plants.     

Molybdate repair of molybdopterin deficient mutants from Chlamydomonas reinhardtii

Summary The phenotypically wild strain I₃ of Chlamydomonas reinhardtii, carrying a cryptic mutation at the nit-6 locus, was distinguished from strains 21gr (cryptic mutant at nit-5) and 6145c (wild type) because of the ability of I₃ to grow on nitrate media containing 2mM tungstate.Molybdopterin-cofactor (MoCo) mutants 102 (double mutant at nit-5 and nit-6) and 104 (mutant at nit-4) grew on nitrate media supplemented with high concentrations of molybdate, although final cell densities were 40–60% lower and generation times 3.5 to six fold longer than for wild type. Under these conditions, nitrate reductase (NR) activity of the mutants, when measured either in situ or in vitro, was practically undetectable. The MoCo defective mutant 307 (nit-3) was not molybdate repairable. Although NR activity was not restored in vitro by molybdate in any of the MoCo^– mutant strains, their extracts had free NR-diaphorase subunits together with NR-subunits assembled into high molecular weight species.Our results indicate that: a) nit-4, nit-5 and nit-6 loci are responsible for molybdate processing in the cell; b) nit-3 may encode a component of the pterin moiety biosynthetic route; c) NR subunits can assemble in the presence of an inactive MoCo; d) high concentrations of molybdate can replace partially in vivo but not in vitro the function of nit-4 and the combined function(s) of the nit-5 and nit-6 gene products.     

Isolation and characterization of dominant,pleiotropic drug-resistance mutants in Chlamydomonas reinhardtii

Summary Three independent pleiotropic drug-resistance (pdr) mutants were isolated by selecting for resistance to the anti-microtubule herbicides amiprophos-methyl (APM) and oryzalin (ORY). These three mutants and a previously isolated mutant, ani1 (anisomycin resistance), were semi-dominant in heterozygous diploids, and they displayed varying degrees of resistance to structurally and functionally unrelated inhibitors such as cycloheximide, cryptopleurine, emetine, atrazine, and nonidet P-40. Linkage analysis and genetic mapping suggested that three of the four mutants, including ani1, define a single locus, here named pdr1. The fourth mutant defined a new locus, pdr2, which is located on the left arm of linkage group VI. One pdr1 mutant exhibited unusual genetic interactions, including enhanced ts-lethality and synergistic increases in drug resistance, when combined with pdr2-1 and with herbicide-resistant alleles of three other genes.     

Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii

Summary Somatic fusion between strains of Chlamydomonas containing complementing cell-wall and auxotrophic mutations, having the same mating-type (mt) 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 (mt ⁺/mt ⁺ or mt ^–/mt ^–) 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 mt ^– 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.     

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

Summary A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.     

Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii

Summary The synthesis of polypeptides related to the CPI chlorophyll-protein complex of photosystem I has been studied by pulse-labeling experiments in twenty chloroplast mutants of Chlamydomonas reinhardtii. Three mutations of the same locus (Girard-Bascou 1987) result in the absence of these CPI-related polypeptides. Among these mutations one, (FUD26) leads to the synthesis of a new polypeptide presumed to be a truncated CPI apoprotein. The molecular characterization of this mutation in the psaA2 gene has been achieved by DNA sequencing the 3 end of this gene. The FUD26 mutation is a 4 base pair deletion resulting in frameshift and premature termination of the protein.     

Direct recombinogenic effect of dimethylnitrosamine (DMN) on zygotes of Chlamydomonas reinhardtii

Summary We developed a test system with mutant strains of Chlamydomonas reinhardtii requiring arginine. Here, the meiotic recombination between two arginine loci served as an indicator of genotoxicity. After crossing, zygotes were treated with the alkylating agent dimethylnitrosamine (DMN) for 30 min. A dose dependent increase of wild type recombinants was found for DMN concentrations ranging from 68 M to 680 M. Like in cells of some higher plants, the agent was active in Chlamydomonas zygotes without any exogenous metabolic activation.     

Changes in chloroplast genome composition and recombination during the maturation of zygospores of Chlamydomonas reinhardtii

Summary In crosses of the unicellular green alga Chlamydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems.     

A new assay system to classify non-mating mutants and to distinguish between vegetative cell and gamete in Chlamydomonas reinhardtii

Summary A new system is described for the classification of various types of non-mating mutants of Chlamydomonas reinhardtii. This system makes use of two simple assays: one for cell body-agglutinin (Saito et al. 1985), and another for the cell wall lytic enzyme (Matsuda et al. 1987). Both assays use the soluble fraction of homogenates from nitrogen-starved cells. Many non-mating mutants previously isolated were analysed and divided into four main phenotypic classes. This assay method is also a valuable tool for distinguishing between vegetative cells and gametes. Without this assay, verifying gametogenesis is especially difficult in agglutinin-and flagella-deficient cells. We found that wild-type cells, which had been grown for 7 days on TAP plates and lacked flagella, contain neither cell body-agglutinin activity nor lytic enzyme activity in the soluble fraction of their cell homogenates, indicating that these cells can be classified as vegetative. When suspended in nitrogen-free liquid medium, the two activities developed rapidly in parallel to the acquisition of mating ability: a maximum level was reached within two hours.     

Behavior of chloroplast genes during the early zygotic divisions of Chlamydomonas reinhardtii

Summary Chloroplast mutations in the green alga Chlamydomonas reinhardtii exhibit a predominantly maternal pattern of inheritance and this pattern can be perturbed by UV irradiation of the maternal gametes prior to mating. In a series of crosses over a range of UV doses, the transmission, segregation, and recombination of mutations at three closely linked chloroplast loci have been examined by pedigree analysis of products arising from the first three post-zygotic divisions. Stocks used in these crosses were constructed to permit identification of the nuclear products of each of the two meiotic divisions and the first post-meiotic mitotic division.A bias toward maternal alleles at all three chloroplast loci was observed in all pedigrees and in zygote clones analyzed from the same crosses many generations after meiosis. This bias decreased with increasing UV dose and with each subsequent division. Segregation of chloroplast genes was rapid during the first three post-zygotic divisions. The type of segregation event from which a given heteroplasmic cell arose had a significant effect on its most likely segregation. pattern in the subsequent division. The results presented here have been discussed in terms of published models of chloroplast gene segregation.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Evidence for persistence of chloroplast markers in the heteroplasmic state in Chlamydomonas reinhardtii

Summary In the green alga Chlamydomonas reinhardtii, reciprocal crosses between strains carrying non-allelic chloroplast mutations to streptomycin dependence (sd-u) produce streptomycin sensitive (sd-u ⁺) recombinant progeny. Transfer of these sd-u ⁺progeny to streptomycin-containing medium results in a much higher frequency of recovery of streptomycin dependent isolates than expected by mutation. Failure to recover the more commonly encountered class of streptomycin resistant mutants also suggests that mutation is not responsible for appearance of the new dependent isolates. Backcrosses of these new sd-u isolates to strains carrying the original sd-u mutations demonstrate their allelism with the sd-u mutation contributed by the mt ⁺parent. Earlier work by Schimmer and Arnold (1969, 1970a-d) indicated that newly isolated sensitive revertants of the streptomycin dependent mutant sd-u-3-18 also yielded high frequencies of sd-u cells but these were never analyzed genetically. We have now obtained new sd-u. isolates from streptomycin sensitive revertants of sd-u-318 and shown them all to be allelic with the original sd-u3-18 mutation. Thus hidden sd-u alleles can coexist with sd-u ⁺alleles in heteroplasmic cells. These heteroplasmic cells are streptomycin sensitive in phenotype and may arise in crosses or from new mutation.     

About biased and non-biased transmission of chloroplast genes following artificial fusion of gametes in Chlamydomonas reinhardtii

Summary Artificial polyethyleneglycol induced fusions of gametes of opposite mating-types carrying chloroplast markers give rise to fusion products transmitting either both markers or the marker from the mt ⁺ or from the mt ^– parent exclusively. The frequencies of the three classes of products were approximately equal in our experiments (Matagne 1981). Similar experiments performed by Matsuda et al. (1983) gave different results, namely a preferential transmission of chloroplast gene from the mt ⁺ parent, very similar to that observed in vegetative zygotes obtained in sexual crosses. Results described here show that in experimental conditions used by Matsuda et al., sexual copulation does occur, leading to formation of zygotes which were misinterpreted as artificial fusion products and gave a biased transmission of chloroplast genes.     

Transmission,recombination and conversion of mitochondrial markers in relation to the mobility of a group I intron in Chlamydomonas

Mitochondrial DNA transmission has been analyzed in diploids produced from sexual crosses or artificial fusions between Chlamydomonas strains which differ by several genetic markers: a group I intron (Cs cob.1 or intron), three restriction sites (Nh, Nc and H markers) located 0.5–5 kb from the insertion site of the intron, and a MUD2 point mutation (27 bp from the insertion site) conferring resistance to myxothiazol. Recombination between mitochondrial markers is a general property of all crosses and fusions analyzed. In crosses between two intron-containing (⁺) strains or two intron-less (^–) strains, the transmission is preferentially paternal (mt ^-), with a preoponderance depending on the nature of the parental genomes. In crosses between (⁺) and (^–) strains, the conversion of intron-less molecules into intron⁺ is frequent when the (⁺) parent is maternal (mt ⁺) and nearly absolute when the (⁺) parent is paternal (mt ^-). In 94% of cases, the conversion is accompanied by the co-conversion of the MUD2 marker. In both crosses and artificial fusions, the conversion of (^–) into (⁺) also influences the transmission of the more distant Nh, Nc and H markers. It is hypothesized that the more frequent transmission of the genome containing the intron results from the elimination of (^–) molecules, as a result of a double-strand cut which is induced by an endonuclease encoded by the intron.     

Detection of chloroplast DNA by using fluorescent monoclonal anti-bromodeoxyuridine antibody and analysis of its fate during zygote formation in Chlamydomonas reinhardtii

Summary A monoclonal anti-bromodeoxyuridine antibody conjugated to fluorescein was used to detect the chloroplast nucleoids after specific incorporation of bromodeoxyuridine (BUdR) into the chloroplast DNA of Chlamydomonas reinhardtii. The incorporation of BUdR was enhanced by simultaneous treatment with fluorodeoxyuridine (FUdR). The method was applied to analyze the fate of chloroplast DNA in zygotes resulting from mating between BUdR-treated gametes (mt ⁺ or mt ^-) and untreated gametes of opposite mating-type. In crosses between wild-type strains, the nucleoids of mt ⁺ origin remained in the large majority of zygotes whereas those of mt ^- origin most often disappeared within the first hours following copulation. In crosses of the type mat-3 mt ⁺xwild-type mt ^- (the mat-3 mutation permits a high transmission of chloroplast genes from the mt ^- parent), the nucleoids of mt ^- origin were generally not eliminated which indicates that the mat-3 mutation prevents the selective destruction of paternal chloroplast DNA in the zygote.     

Transmission of chloroplast genes in crosses between Chlamydomonas reinhardtii diploids: Correlation with chloroplast nucleoid behavior in young zygotes

Summary In crosses between diploid strains of Chlamydomonas reinhardtii, the mode of chloroplast gene transmission was studied and correlated with the behavior of DAPI-stained chloroplast nucleoids in young tetraploid zygotes. Phenotypically plus (female) diploids with mating-type genotypes of mt ⁺/mt ⁺ or mt ⁺/0 (hemizygote) and phenotypically minus (male) diploids with genotypes of mt ⁺/mt ^– or mt ^–/mt ^– or 0/mt ^– were obtained by either crosses or polyethylene glycol induced somatic fusions between haploid strains. When crosses were made between mt ⁺/mt ⁺ or mt ⁺/0 and mt ^–/mt ^– or 0/mt ^–, the transmission of chloroplast genes occurred mostly from the female parent, indicating a typical maternal inheritance. In these cases the chloroplast nucleoids of one of the two parents disappeared within 2.5 h after mating. In contrast, when female diploids were crossed to mt ⁺/mt ^– male diploids, the chloroplast genes from both parents were transmitted to the progeny at equal frequency. The chloroplast nucleoids derived from the two parents were conserved for at least 2.5 h after mating. It is concluded that the chloroplast genes derived from the heterozygous male (mt ⁺/mt ^–) are protected from the degradation that takes place soon after zygote formation, by virtue of the presence of mt ⁺ gene although this allele is recessive with respect to the phenotypic expression of sexuality in diploids.