Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.     

Disappearance of the heteroplasmic state for chloroplast markers in zygospores of Chlamydomonas reinhardtii

In the investigations reported here, the length of zygospore incubation or “maturation” prior to the induction of meiosis was found to affect the inheritance pattern of chloroplast genes. The frequency of zygospores transmitting chloroplast alleles from both parents drops with increasing zygospore age following mating, while the frequencies of zygospores homoplasmic for maternal or paternal chloroplast alleles increase correspondingly. Since there is a negligible reduction in viability, zygospores which are initially biparental appear to become pure for the chloroplast genes from one or the other parent prior to the occurrence of cell division. These results are amplified in crosses of mt⁺ cells which have been irradiated with ultraviolet (uv) light or grown in the presence of the base analog, 5-fluorodeoxyuridine, which also perturbs maternal inheritance. Low doses of uv irradiation, applied to zygospores derived from crosses in which the maternal parent was also irradiated prior to mating, increase the biparental zygospore frequency while reducing the proportion of maternal zygospores. This indicates that at least some maternal zygospore clones are actually derived from zygospores which still contain both parental chloroplast genomes prior to the induction of germination. Thus, a subclass of zygospores must contain paternal chloroplast genomes which are either eliminated upon germination or are not expressed in the resulting zygospore clone. Tetrad analysis of biparental zygospores derived from uv-irradiated mt⁺ gametes demonstrates that the frequency of maternal chloroplast alleles in biparental zygospores decreases as they age. One result is an increase in the proportion of meiotic products homoplasmic for all paternal markers. The increased segregation of homoplasmic daughter cells during the meiotic divisions may result from a reduction in chloroplast ploidy by elimination of maternal genomes. Alternatively, it may reflect an altered ratio of maternal:paternal genomes due to continuous rounds of pairing and gene conversion between heterologous chloroplast DNAs leading to genetic drift within the DNA population of the organelle.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

Stable chloroplast transformation in Chlamydomonas reinhardtii using microprojectile bombardment

The chloroplasts ofChlamydomonas reinhardtii were transformed using a vector (paadAGUS4.1) that contained a spectinomycin-resistance gene (aadA) as a selectable gene, and bacterialuidA (GUS) as a reporter gene, and pea 4.1 kb D-loop containing sequence. The vector was introduced into the alga through particle gun bombardment. The transformed colonies were screened for the presence of foreign genes by Southern hybridization using GUS,aadA and 4.1 pea Ori probes. Expression ofaadA and GUS genes was detected in all colonies that were grown on spectinomycin. A detailed restriction analysis followed by southern hybridization of total genomic DNA using pea 4.1 kb D-loop as probe indicated that the D-loop sequence can serve in site-specific integration of foreign DNA due to high homology. Restriction analysis of different colonies showed that the foreign DNA was probably present in a mixture population of autonomous segment and integrated in the native chloroplast genome.