Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

The pattern of amyloplast DNA accumulation during wheat endosperm development

The accumulation of amyloplast DNA during endosperm development was studied in two cultivars of spring wheat, Triticum aestivum L. Chinese Spring (CS) and Spica, small and relatively larger-grained cultivars, respectively. Endosperms were isolated between 9 and 45 days post anthesis (dpa) and the amyloplast DNA content of endosperm nucleic-acid extracts was measured by quantitative hybridisation with a homologous chloroplast-DNA probe. The endosperm cells of CS and Spica accumulated amyloplast DNA during development in a similar way. In both cultivars there was a large increase in the amount of plastid DNA (ptDNA) per endosperm between 9 and about 15 dpa, after which there was no further increase. Because nuclear DNA continued to accumulate until 24 dpa, the percentage contribution of amyloplast DNA to total DNA fluctuated in both cultivars during development, reaching maxima at 12 dpa of about 1.00% and 0.85%, and dropping to apparently constant levels of 0.60% and 0.52% in CS and Spica, respectively, by 24 dpa. In both cultivars, the average number of ptDNA copies per amyloplast was calculated to increase from about 10 copies at 9 dpa to about 50 copies in the mature amyloplasts at 31 dpa. However, the heavier endosperms of Spica contain more cells than those of CS and the varieties therefore differed in the amount of ptDNA that accumulated per endosperm: Spica endosperms accumulated 110 ng of ptDNA by 15 dpa, compared with only 85 ng in CS. The apparent accumulation of ptDNA copies in wheat amyloplasts during endosperm development contrasts with the decline in chloroplast-DNA copies in wheat chloroplasts during leaf development.Abbreviations CS Chinese Spring - ctDNA chloroplast DNA - dpa days post anthesis - kbp 10³ base pairs - nDNA nuclear DNA - ptDNA plastid DNA - mtDNA mitochondrial DNA     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

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.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Nuclear DNA content of ploidy chimeras of Hosta Tratt. (Hostaceae) demonstrate three apical layers in all organs, but not in the adventitious root

A unique combination of chloroplast and ploidy chimeras is found in some Hosta Tratt. (Hostaceae) cultivars. The absence of endopolyploidy in the above–ground parts made it possible to study the fate of the apical or germ layers in all organs. Moreover, we would like to address the question of the number of apical cell layers in Hosta also to explain breeding results. Nuclear DNA content in twelve different organs of Hosta was determined by flow cytometric analysis of four chimeric cultivars with a 4-2-2 or 2-2-4 composition. The margin of the leaf, derived from the L1, can be determined separately and has a 4C amount of DNA in 4-2-2 ploidy chimeras but a 2C amount of DNA in 2-2-4 chimeras. The presence of three layers in Hosta was deduced from comparisons of the percentage of 4C nuclei in (2)-2-4 and (4)-2-2 chimeras. If Hosta organs derive only from two layers the percentage of 4C cells in the different ploidy chimeras should add up to 100%, but they do not. The absence of nuclei with a 2C amount of DNA in the roots of 2-2-4 plants shows that only the L3 participates in adventitious root formation. Gametes and consequently seedlings are mainly derived from the L2. The often found correlation between the color of the centre of the leaf and the color of the seedlings is the consequence of the way these cultivars originate. The presence of three apical layers is visualized in the tricolored leaves of Hosta cultivars like `Striptease'. The nuclear DNA amounts show unequivocally, that in all the above ground parts of Hosta the three apical layers L1, L2 and L3 are present. However, only the L3 participates in adventitious root formation.     

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.     

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance

Summary The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F₁-hybrids.     

Sequence homology to the Drosophila per locus in higher plant nuclear DNA and in Acetabularia chloroplast DNA

Summary In plant cells a DNA sequence was found which is homologous to the Drosophila per locus. In rape and spinach the homologous sequence occurs in the nuclear but not in the chloroplast genome while in Acetabularia it is found in the chloroplast but not in the nuclear genome. A 1.175 kb EcoRI-SalI fragment of the chloroplast genome of Acetabularia containing the homologous sequence was subcloned into pUC12 and sequenced. The core of the 1.175 kb fragment is a repetitive tandemly arranged sequence of 43 units of the hexamer GGA ACT coding for glycine and threonine.Abbreviations MES N-morpholinoethanesulfonic acid - DTE dithioerythritol - DTT dithiothreitol - nDNA nuclear DNA - ctDNA chloroplast DNA - TEP Tris, EDTA, proteinase K buffer     

Cultivated potato chloroplast DNA differs from the wild type by one deletion — evidence and implications

Summary The chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

Characterization of the nuclear DNA ofPhialophora gregata ff.sp.adzukicola andsojae

Phialophora gregata nuclear (n) DNA was characterized by physical methods. The nDNA of f.sp.adzukicola was shown to be larger than that of f.sp.sojae, 2.9 and 2.1 × 10¹⁰ Da, respectively. The amounts of repetitive sequence and AT-rich region in the nDNA were also larger in f.sp.adzukicola than f.sp.sojae. These results indicate that the nuclear genome organization of the two formae speciales is differentiated.     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

Cytoplasmic male sterility and nuclear restorer genes in a natural population of Beta maritima: genetical and molecular aspects

Summary One natural population (F₀ generation) of Beta maritima situated on the French Atlantic coast has been analysed. It was composed of 62% female, 30% hermaphrodite and 8% intermediate plants. The analysis of half-sib progeny (F₁ generation) obtained from in situ open pollination demonstrates the cytoplasmic determination of male sterility in Beta maritima and the restoration of fertility by nuclear genes. The mitochondrial DNA (mtDNA) and the chloroplast DNA (ctDNA) of sixteen F₁ plants, extracted from offspring of the three sexual phenotypes, were analysed using the restriction enzymes Sal I and Bam HI, respectively. Two cytoplasmic lines with their own peculiar genetic characteristics were distinguished using the restriction enzyme patterns of mtDNA: (i) the S cytoplasmic line was found in segregating progeny of two F₀ plants; all three phenotypes were produced (that is, progeny including hermaphrodite, female and intermediate plants); (ii) the N cytoplasmic line was found in the progeny of one F₀ hermaphrodite plant; this produced only hermaphrodites. Thus, segregating and non-segregating hermaphrodite F₀ plants can be distinguished. The nuclear genes maintaining sterility or restoring fertility are expressed in line S. At the same time the analysis of Beta vulgaris material has been carried out at the molecular level: N cytoplasmic lines of B. vulgaris and B. maritima differed only by 3 fragments of mtDNA; but the S cytoplasmic line of B. maritima was very different from Owen's cytoplasmic male sterile line of B. vulgaris. No variation in the ctDNA pattern was detected within and between the two taxa.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

Summary The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.     

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.