Chronological transition of mitochondrial morphology in Chlamydomonas reinhardtii (Chlorophyceae) poststationary phase growth1

In Chlamydomonas reinhardtii P. A. Dangeard, mitochondrial morphology has been observed during asexual cell division cycle, gamete and zygote formation, zygote maturation, and meiotic stages. However, the chronological transition of mitochondrial morphology after the stationary phase of vegetative growth, defined as the poststationary phase, remains unknown. Here, we examined the mitochondrial morphology in cells cultured for 4 months on agar plates to study mitochondrial dynamics in the poststationary phase. Fluorescence microscopy showed that the intricate thread‐like structure of mitochondria gradually changed into a granular structure via fragmentation after the stationary phase in cultures of about 1 week of age. The number of mitochondrial nucleoids decreased from about 30 per cell at 1 week to about five per cell after 4 months of culture. The mitochondrial oxygen consumption decreased exponentially, but the mitochondria retained their membrane potential. The total quantity of mitochondrial DNA (mtDNA) of cells at 4 months decreased to 20% of that at 1 week. However, the mitochondrial genomic DNA length was unchanged, as intermediate lengths were not detected. In cells in which the total mtDNA amount was reduced artificially to 16% after treatment with 5‐fluoro‐2‐deoxyuridine (FdUrd) for 1 week, the mitochondria remained as thread‐like structures. The oxygen consumption rate of these cells corresponded to that of untreated cells at 1 week of culture. This suggests that a decrease in mtDNA does not directly induce the fragmentation of mitochondria. The results suggest that during the late poststationary phase, mitochondria converge to a minimum unit of a granular structure with a mitochondrial nucleoid.     

Organellar DNA replication inNicotiana tabacum cultured cells

In the diploid vegetative plant cell, the nuclear DNA is present in two copies, whereas the chloroplast and mitochondria genomes are present in a higher and variable copy number. We have studied the replication of the nuclear, chloroplast and mitochondrial DNA in culturedNicotiana tabacum cells using density and radioactive markers. Essentially all the 10 000 chloroplast genomes in a given cell replicate in one cell cycle as do all the mitochondrial DNA molecules. No measurable level of unreplicated organellar DNA molecules can be detected in these cells.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Nuclear and mitochondrial DNA have sequence homology with a chloroplast gene

Summary A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P₇₀₀ chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.     

Natural hybridization of Yezo and Sakhalin spruce in central Hokkaido,revealed by DNA markers with contrasting modes of inheritance

Yezo spruce (Picea jezoensis var. jezoensis) and Sakhalin spruce (Picea glehnii) occur across Hokkaido and co‐occur in some forest habitats. This leads to the potential for natural hybridization between these two species, which has been shown to occur at low frequencies. The purpose of this study was to identify these hybrids and their possible mating patterns, using various Pinaceae DNA markers with different modes of inheritance. The markers used were maternally inherited mitochondrial DNA (mtDNA), paternally inherited chloroplast DNA (cpDNA) and biparentally inherited nuclear microsatellites (nSSRs). Seven putative natural hybrids, four artificially‐crossed F₁ hybrids, four parent plants from each species, and two artificially‐backcrossed hybrids of putative natural hybrids and their parents were analyzed using the diagnostic DNA markers developed in this study. We found Yezo spruce and Sakhalin spruce to be distinct (J and G types, respectively), and the modes of inheritance held true for the two species, as was previously reported to be the case in Pinaceae. Four of the seven putative natural hybrids harbored J‐type cpDNA, G‐type mtDNA and J/G‐type nSSRs, indicating that natural F₁ hybrids are likely to arise from a G (female) × J (male) crossing. One natural hybrid harbored G‐type cpDNA, J‐type mtDNA and J/G‐type nSSRs, which implies that hybrids produced by J (female) × G (male) crossings occur at low frequencies. The two remaining hybrids harbored J‐type cpDNA and mtDNA with either J/G or J/J‐type nSSRs, suggesting that they may be F₂ hybrids resulting from backcrossing between an F₁ hybrid and a Yezo spruce.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

Summary Synchronous cultures of the algaDunaliella salina were grown in blue or red light. The relationships between replication of chloroplast DNA, cell size, cell age and the number of chloroplast nucleoids were studied. The replication of chloroplast DNA and the division of chloroplast nucleoids occurred in two separate periods of the chloroplast cycle. DNA replication was concomitant with that in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown cells were bigger and grew more rapidly than those grown in blue light. In newly formed daughter cells, the chloroplast nucleoids were small and spherical and they were localized around the pyrenoid. During the cell cycle they spread to other parts of the chloroplast. The number of DNA molecules per nucleoid doubled during DNA replication in the first third of the cell cycle but decreased several hours later when the nucleoids divided. Their number was fairly constant independent of the different light quality. Cells grown in red light replicated their chl-DNA and divided their nucleoids before those grown in blue light and their daughter cells possessed about 25 nucleoids as opposed to 15.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15–20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per g of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.     

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the Moss Physcomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

Chloroplast DNA‐based studies in molecular ecology may be compromised by nuclear‐encoded plastid sequence

Ongoing genetic transfer from mitochondria and plastids into the nucleus is a well‐documented fact. While in metazoan molecular ecology the need for surveillance against pseudogene‐mediated artefacts when analysing mtDNA sequences is commonly accepted, no comparable measurements have been established for plastid‐based studies. We highlight the impact and management of nuclear mitochondrial insertions, argue that nuclear plastid sequences represent an underestimated but major factor in plant molecular ecology, and discuss potential avenues of remedy in chloroplast studies.     

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.