Fluorescence microscopy of DNA-protein structures from osmotically lysed mitochondria of yeast and tobacco

Summary The mitochondrial nucleoid is a compact structure composed of DNA and protein. By fluorescence microscopy, decondensation of the nucleoids was observed when yeast and tobacco mitochondria were osmotically lysed and subjected to an electric field. Structures stained with ethidium bromide were seen moving toward either the anode or the cathode. Since the movement of deproteinized DNA is toward the anode, the structures moving toward the cathode represent DNA-protein complexes with a net positive charge. Nucleoid decondensation and unfolding of the DNA probably resulted from the removal of weakly bound proteins; yet high-affinity basic proteins were evidently retained yielding cationic DNA-protein structures. Some of the positively charged structures were observed to break, presumably at single-stranded DNA regions, releasing negatively charged particles. The DNA-protein structures were complex branching forms larger than the unit genome, suggesting that multigenomic, concatemeric DNA is present within the mitochondria.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EtBr ethidium bromide - HMG high-mobility group - mt-genome mitochondrial genome - mt-nucleoid mitochondrial nucleoid - PFGE pulsed-field gel electrophoresis - pt-nucleoid plastid nucleoid - ssDNA single-stranded DNA     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize

We examined the DNA from chloroplasts obtained from young and fully expanded leaves of tobacco (Nicotiana tabacum L.), Medicago truncatula, pea (Pisum sativum L.), and maize (Zea mays L.). The changes in plastid DNA content and structure were monitored by four independent methods: 4′,6-diamidino-2-phenylindole (DAPI) staining with intact chloroplasts, in situ DAPI staining of cytological sections, ethidium bromide staining at the single-molecule level after exhaustive deproteinization of lysed chloroplasts, and pulsed-field gel electrophoresis. During leaf development, we found a decline of chloroplast DNA (cpDNA) in all four plants. For tobacco, for which plants can readily be regenerated from somatic cells, cpDNA persisted longer than in the other three plants. We also found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules during plastid development. Although the decrease in molecular size and complexity paralleled the decrease in DNA content per plastid, 6% of the chloroplasts in a fully expanded tobacco leaf still contained DNA in complex branched structure, whereas no such complex structures were found in mature leaves for the hard-to-regenerate maize.     

Imaging of organelles by electron microscopy reveals protein-protein interactions in mitochondria and chloroplasts

Ongoing progress in electron microscopy (EM) offers now an opening to visualize cells at the nanoscale by cryo-electron tomography (ET). Large protein complexes can be resolved at near-atomic resolution by single particle averaging. Some examples from mitochondria and chloroplasts illustrate the possibilities with an emphasis on the membrane organization. Cryo-ET performed on non-chemically fixed, unstained, ice-embedded material can visualize specific large membrane protein complexes. In combination with averaging methods, 3D structures were calculated of mitochondrial ATP synthase at 6 nm resolution and of chloroplast photosystem II at 3.5 nm.     

High levels of population differentiation for mitochondrial DNA haplotypes inPinus radiata,muricata, andattenuata

We analyzed mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs) associated with cytochrome oxidase, subunit I (coxI)-related gene sequences in 268 trees derived from 19 natural populations of three species of pines from California (USA): Monterey pine (Pinus radiata D. Don), bishop pine (P. Muricata D. Don), and knobcone pine (P. attenuata Lemm.). Total genomic DNA was digested with four restriction endonucleases and probed with a 750-bp fragment of the mitochondrialcoxI gene amplified fromP. attenuata via the polymerase chain reaction (PCR). ThecoxI gene is repeated at least 4 times in some populations, and all variants that we observed resulted from complex rearrangements rather than from point mutations. There was limited intrapopulation variation, but strong differentiation among populations. When applied to haplotype frequencies, Nei's gene diversity within populations (H_s) averaged 7% (±3), and G_st varied from 75% forP. Radiata to 96% forP. muricata. The high degree of population differentiation for mtDNA suggests that it can be a powerful marker of population differences, but its rapid rate of structural evolution appears to result from recombination among a limited number of repetitive elements-giving frequent homoplasious fragment phenotypes. The phylogenetic trees disagreed with results from chloroplast DNA, nuclear gene, and morphological studies.     

Localization of plasmidlike DNA in giant-celled marine green algae

Summary Novel extrachromosomal DNA molecules were localized in giant-celled marine green algae by organelle isolation and fluorescence in situ hybridization methodologies. Nucleic acids extracted from isolated chloroplasts ofErnodesmis verticillata andVentricaria ventricosa were greatly enriched in plasmidlike DNA species. Fluorescence in situ hybridization was employed to resolve further the subcellular location of these molecules. Cloned restriction fragments of the algal plasmidlike DNA hybridized solely to low-molecular-weight DNA in Southern blots; they did not hybridize to any chromosomal DNA. Probes were generated from these clones that either did (Northern-positive) or did not (Northern-negative) hybridize to RNA species in Northern blots. Probes specific for localizing the plasmidlike DNA were generated from the latter clones, whereas probes potentially localizing both DNA and relevant mRNA species were generated from the former ones. After hybridization and signal amplification via indirect immunofluorescence, fluorescent punctae were visible surrounding the single pyrenoid in each chloroplast with both types of probes. The punctae were arranged in a hollow spherical configuration, as resolved by confocal laser scanning microscopy. Nearly twice as many punctae per chloroplast were present inV. ventricosa (11.5) as there were inE. verticillata (6.0). The differential distribution of plasmidlike DNA within each chloroplast was in contrast to chloroplast chromosomal DNA, which occurred as multiple nucleoids scattered throughout the entire organelle. The localization of plasmidlike DNA within chloroplasts correlates well with previous sequence data indicating that these molecules contain putative open reading frames encoding protein components of photosystems I and II.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylmdole - FITC fluorescein isothiocyanate - FISH fluorescence in situ hybridization - HMW high molecular weight - LMW low molecular weight - ORF open reading frame     

Organelle DNA polymorphism in apple cultivars and rootstocks

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect chloroplast (cp) and mitochondrial (mt) DNA variation among 18 apple cultivars and three rootstocks. The distribution of RFLP patterns allowed the assignment of these genotypes into three groups of cytoplasmic relatedness. Our results also demonstrate maternal inheritance of cp- and mtDNAs in apple. Thus, the organelle DNA assay provides a convenient and reliable method to assess cytoplasmic diversity within the apple germ-plasm collection and to trace the maternal lineages involved in the evolution of apple.     

Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in loblolly pine

Summary The inheritance of organelle DNAs in loblolly pine was studied by using restriction fragment length polymorphisms. Chloroplast DNA from loblolly pine is paternally inherited in pitch pine x loblolly pine hybrids. Mitochondrial DNA is maternally inherited in loblolly pine crosses. The uniparental inheritance of organelle genomes from opposite sexes within the same plant appears to be unique among those higher plants that have been tested and indicates that loblolly pine, and possibly other conifers, must have special mechanisms for organelle exclusion or degradation or both. This genetic system creates an exceptional opportunity for the study of maternal and paternal genetic lineages within a single species.     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Reconstitution of 50 S ribosomal subunits from Bacillus stearothermophilus with 5 S RNA from spinach chloroplasts and low-Mr RNA from mitochondria of Locusta migratoria and bovine liver

Reconstitution experiments with 50 S ribosomal subunits from Bacillus stearothermophilus demonstrate that spinach chloroplast 5 S rRNA can be incorporated into the bacterial ribosome and yield biologically active particles, thereby establishing the eubacterial nature of chloroplast 5 S rRNA. In contrast, mitochondria from Locusta migratoria or bovine liver do not appear to contain discrete, low-Mr RNAs, which can replace 5 S rRNA in the functional reconstitution of B. stearothermophilus ribosomes.     

Connection between the rate of cooling and fluorescence properties at 77 K of isolated chloroplasts

Cooling of chloroplasts to ?196°C can under certain circumstances lead to an erroneous analysis of energy distribution. After minimizing influences of sample geometry and effects of plastid concentration it is shown that externally induced membrane change leads to an increase in the ratio $\text{F}{}_{740}\text{F}{}_{687}$ of the fluorescence emission spectrum. Similar alterations can be observed by variation of the rate of cooling the plastids to 77 K, especially if whole chloroplasts are used. The differences in emission ratios are indicative also of changes in initial energy distribution between the photosystems, given here by the value α_N. This is inferred from experiments with either osmotically induced thylakoid disturbances or those effected through a slow cooling process. The circumstances and the significance of these observations are discussed.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Monokaryotic chloroplast mutation has no effect on non-Mendelian transmission of chloroplast and mitochondrial DNA in Chlamydomonas species

Summary. We studied whether the monokaryotic chloroplast (moc) mutation affects the transmission of chloroplast and mitochondrial DNA in Chlamydomonas species. We used a previously isolated moc mutant from our cell line G33, which had only one large chloroplast nucleus. To obtain zygotes we crossed the mutant cells with wild-type cells, and mutant cells with receptive mates (females [mt+] with males [mt–]). In these zygotes, we recorded preferential dissolution of mt– parental chloroplast nuclei and fusion of the two cell nuclei. Antibiotic-resistance markers of chloroplast DNA were maternally transmitted in all crosses. PCR analysis of the cytochrome b (cob) gene sequence showed that the mitochondrial DNA was paternally transmitted to offspring. These results suggest that the moc mutation did not affect the organelle DNA transmission.Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan.     

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.     

Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype

We examined the chloroplast DNA (cpDNA) from plastids obtained from wild type maize (Zea mays L.) seedlings grown under different light conditions and from photosynthetic mutants grown under white light. The cpDNA was evaluated by real-time quantitative PCR, quantitative DNA fluorescence, and blot-hybridization following pulsed-field gel electrophoresis. The amount of DNA per plastid in light-grown seedlings declines greatly from stalk to leaf blade during proplastid-to-chloroplast development, and this decline is due to cpDNA degradation. In contrast, during proplastid-to-etioplast development in the dark, the cpDNA levels increase from the stalk to the blade. Our results suggest that DNA replication continues in the etioplasts of the upper regions of the stalk and in the leaves. The cpDNA level decreases rapidly, however, after dark-grown seedlings are transferred to light and the etioplasts develop into photosynthetically active chloroplasts. Light, therefore, triggers the degradation of DNA in maize chloroplasts. The cpDNA is retained in the leaf blade of seedlings grown under red, but not blue light. We suggest that light signaling pathways are involved in mediating cpDNA levels, and that red light promotes replication and inhibits degradation and blue light promotes degradation. For five of nine photosynthetic mutants, cpDNA levels in expanded leaves are higher than in wild type, indicating that nuclear genotype can affect the loss or retention of cpDNA.