Mitochondrial genetics of Chlamydomonas reinhardtii: resistance mutations marking the cytochrome b gene.

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.

A method for complementation analysis of nuclear and chloroplast mutants of photosynthesis in chlamydomonas.

The photosynthetic properties of young zygotes of Chlamydomonas reinhardi were analyzed. In heterozygotes for two nuclear or two chloroplast mutations affecting photosynthesis, recovery of photosynthetic activity was observed that is most likely the result of intergenic complementation.--We observed that chloramphenicol inhibited the recovery of activity in double heterozygotes for mutants lacking at least one thylakoid polypeptide of chloroplast origin, while it had not effect on wild-type homozygotes. This indicates that the recovery of activity in double heterozygotes could result from the repair of existing thylakoid membranes by de novo synthesis of the missing polypeptides.

Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii.

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria.

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol.

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.

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction.

A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.

Etiolated cells of Chlamydomonas reinhardtii: choice material for characterization of mitochondrial membrane polypeptides.

We have investigated a light-conditional mutant of Chlamydomonas reinhardtii (J12) that is unable to synthesize chlorophyll in the dark with the aim of characterizing the mitochondrial membrane polypeptides of this alga. A crude membrane fraction derived from etiolated cells was analyzed by gel electrophoresis, immunoblot analysis, and pulse-labeling in the presence of specific protein synthesis inhibitors. This fraction contained both mitochondrial and etioplast membranes, and the latter contained appreciable amounts of subunits of the cytochrome b6f complex. The mitochondria-encoded subunit 1 of cytochrome-c oxidase called COX1 was identified, and its synthesis was detected in this membrane fraction. The redox-difference spectra of mitochondrial cytochromes were studied in whole cells and membrane fractions, in both respiratory-competent and -deficient strains. Mitochondrial membranes could be further purified after sucrose gradient centrifugation. The use of etiolated cells and their membrane extracts, in association with appropriate methodologies, opens ways to study the molecular genetics of mitochondria in C. reinhardtii and allows us to address the question of the cooperation established between the three genetic compartments of a plant cell.

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

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.

Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii.

Genetic analysis of 25 nuclear mutants defective in the chlorophyll-protein complex CP1 was undertaken. The mutants belong to 13 complementation groups scattered throughout the nuclear genome. All these mutants lack the apoprotein of CP1 and, in addition, a specific set of six low molecular weight thylakoid polypeptides. System I particles obtained by treating WT thylakoid membranes with detergent specifically contain those polypeptides which the mutants lack. These observations suggest that a particular sub-structure of the thylakoid membrane associated with the photosystem I activity is missing from all 25 mutants studied, and that this general phenotype can result from mutation at any one of several unlinked Mendelian loci.

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene.

We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.