The plastome mutator of Oenothera continues to function as originally described

Summary Recently, Lindenhahn et al. (1985) hypothesized that the plastome mutator (pm) system in Oenothera originated through contaiminating cross-pollination and that the variegation was an example of hybrid plastome-genome incompatibility. Their evidence was based on restriction pattern analyses of white sectors which showed wild-type plastome III patterns rather than the wild-type plastome I patterns of the green portions of their plants. Their hypothesis does not adequately account for the results which our laboratories have obtained independently; the pm-system of Oenothera continues to generate many new and different plastome mutations following the genetic parameters as published originally (Epp 1973). Our studies support mutator gene function. The restriction pattern of the chloroplast DNA of five newly isolated pm-induced variegation sectors are reported here to show a restriction pattern identical to the green wild-type plastids. The restriction pattern reported by Lindenhahn et al. (1985) for their white sector plastids is different than we would expect from a pm-induced plastome mutation. Their overall analysis did not utilize many of the salient features of the genetics of Oenothera and of the pm-system. The white sectors they observed are probably due to an accidental contamination by plastome III plastids. Suggestions are made for delineating experimentally plastome mutations and hybrid incompatibility. For future analyses, a comparative study of numerous pm-induced sectors is recommended, since the pm-system readily generates many different plastome mutations with independent origins. This comparison would greatly assist in the interpretation of restriction patterns.     

Oenothera chloroplast DNA polymorphisms associated with plastome mutator activity

Molecular Genetics and Genomics - Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than...     

The Oenothera plastome mutator: effect of UV irradiation and nitroso-methyl urea on mutation frequencies

Summary Oenothera plants homozygous for a recessive plastome mutator allele (pm) showed spontaneous mutation frequencies for plastome genes that are 200-fold higher than spontaneous levels. Mutations occurred at high frequencies in plants grown in the field, in a glass-house, or as leaf tip cultures under fluorescent light, indicating that the plastome mutator activity is UV-independent. However, the chlorotic sectors became visible at an earlier stage of development when seedlings were irradiated, compared to seedlings that were not exposed to UV. These results imply that the rate of sorting-out was increased by the irradiation treatment, possibly due to a decrease in the effective number of multiplication-competent plastids, or a reduction in the extent of cytoplasmic mixing. Nitroso-methyl urea treatment of seeds had a dramatic effect on mutation frequency in both wild-type and plastome mutator samples. When the background mutation rates were low, the combination of the plastome mutator nucleus and the chemical mutagenesis treatment resulted in a synergistic effect, suggesting that the plastome mutator may involve a cpDNA repair pathway.     

The falcifolia syndrome of Oenothera: IV. Loss of falcifolia-determining factors

Summary Inheritance of the falcifolia syndrome is explained by the hypothesis that this developmental disturbance is caused by the interaction of a non-chromosomal determinant (sigma) of Oenothera suaveolens with a complementary determinant (lambda) of O. lamarckiana when these two species are crossed. Localization of the determinants in the cell, the mechanism of their expression, and their molecular nature have not been established. Somatic segregation within the hybrids may lead to a separation of the two components, as suggested by the different reactions of sister plants in backcross tests and on selfing. The present paper presents results showing that in rare cases the fal determinants may be eliminated from the hybrid's cytoplasm. Thus, plants are produced that are phenotypically identical with the original parental strains but in crosses react like species that lack such determinants.     

The falcifolia syndrome of Oenothera: III. The general pattern of its non-Mendelian inheritance

Summary The falcifolia (fal) syndrome is a malformation characterized by shoot sectors with sickle-shaped leaves, which appears in hybrids between Oenothera suaveolens and O. lamarckiana and shows a non-chromosomal inheritance of a previously undescribed type. The determinants, their location in the cell, and the mechanism of their expression are unknown. The defect is the result of a cross in which mixing of two different cytoplasms occurs, without the usual predominantly maternal inheritance. F₁ progeny of reciprocal crosses show a quantitative difference in the frequency and degree of expression of the fal character. When the F₁ progeny are backcrossed to the parents, the percentage of fal is high in crosses to O. suaveolens and low in those to O. lamarckiana. This manner of transmission is observed regardless of whether the hybrid is used as seed or pollen parent or shows a normal or fal phenotype. F₂ generations from F₁ plants having either a normal or a fal phenotype always include a certain percentage of fal plants, although the latter generally produce a higher percentage of fal progeny. If a second backcross is carried out, plants that produce normal progeny on self-pollination behave differently from those that produce some fal off-spring when selfed. The latter are similar to the F₁ with regard to the transmission of the fal trait. Plants of the F₁B₁ yielding normal progeny upon selfing produce normal progeny in the F₁B₂ if the parent to which they are backcrossed is the same as in the first backcross; if the parents of the first and second backcross differ, a high percentage of fal offspring is obtained. Again, whether the hybrid is used as seed or pollen parent is not relevant. Exceptions to this behaviour have been observed only rarely in that the character of the penultimate cross is retained. There is some evidence of somatic segregation of the fal determinants, since sister plants may react differently; this suggestion is supported by comparing the progenies of different branches of a self-pollinated fal plant of the F₁ generation.Abbreviations F₁, F₂, F₃, F₄ First through fourth filial generation, obtained by self-pollination - F₁B₁ First backcross generation, i.e. the F₁ was backcrossed to one of the original parents - F₁B₂ Second backcross generation, i.e. the F₁B₁ was backcrossed to one of the original parents - F₁B₃ Third backcross generation, i.e. the F₁B₂ was backcrossed to one of the original parents - (F₁B₁)D₁ Descendants obtained by self-pollination of a F₁B₁ plant; further generations obtained by self-pollination are designated as D₂, D₃, D₄ - (F₁B₁)D₁B₁ Descendant or generation obtained by a backcross of the D₁ of an F₁B₁. Backcrosses of the D₂ and D₃ are designated mutatis mutandis - (F₁B₁)D₁B₂ Generation obtained by a backcross of the (F₁B₁)D₁B₁     

Influence of ploidy on plastome mutagenesis in Nicotiana

Summary A clear influence of ploidy was observed on the frequency of both spontaneous and nitroso-methylurea (NMU) induced, streptomycin-resistant, adventitious shoots developing on leaf explants of Nicotiana tabacum and N. plumbaginifolia. At nearly all NMU levels employed a significantly higher yield of resistant shoots was obtained from haploid compared with diploid leaf strips. At 1 mM NMU the differences were not significant and were absent when a high (1000 mg/1) selective concentration of streptomycin sulphate was used. The influence of ploidy is discussed in relation to the possible effect of plastome copy number on mutagenesis and sorting out of resistant plastids.     

Site-specific circularisation at an intragenic sequence in Oenothera mitochondria

Summary The mitochondrial genome of Oenothera is divided between a number of circular molecules that are derived from master circles containing the total sequence complexity. The circularisation event which leads to the formation of one of the small molecules involves a decanucleotide within the region coding for the large ribosomal RNA. This decanucleotide, 5-GGAAGCAGCC-3, is repeated 7.5 kb further downstream. The repeat recombines with the intragenic sequence to form circle no. 3, which thus contains the 3 part of the large rRNA gene. The other theoretical result from such a circularisation with the 5 part of the gene cannot be detected in the mitochondrial genome, thus eliminating the reformation of the original sequence arrangement. Different models are discussed for this apparent non-reciprocal circularisation.     

Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa including P. vulgaris and P. coccineus

The genetic variability of seven Phaseolus taxa has been evaluated on the basis of molecular data and the results have used to clarify the phyletic relationships between several taxa of the P. coccineus L. complex. Chloroplast DNA (cpDNA) from 33 populations was digested with six restriction endonucleases, revealing some polymorphisms that made it possible to divide most of the taxa into two main groups: the subspecies of P. coccineus on the one hand, and P. vulgaris L., P. polyanthus Greenman and P. costaricensis (Freytag and Debouck) on the other hand. P. polyanthus is closer to P. vulgaris than the other taxa of the second group and should be considered as a separate species. The position of the wild species P. costaricensis is intermediate between P. coccineus and P. polyanthus. P. glabellus shows sufficient polymorphisms at the cpDNA level to be recognized as a separate species, as previously suggested from total seed-protein electrophoretic studies. These results favour the hypothesis of a common phylogeny for P. vulgaris, P. polyanthus, P. costaricensis and P. coccineus from a single wild ancestor. Although cpDNA is generally known to be uniform at the intraspecific level, some additional polymorphisms were also detected within P. vulgaris, P. polyanthus and P. coccineus. Further studies are required to understand the significance of the latter.     

The Cucumis plastome: physical map,intrageneric variation and phylogenetic relationships

Summary A restriction map of the Cucumis melo L. (melon) plastome was constructed by using several mapping approaches: single and double digestions of the chloroplast DNA (chlDNA) with endonucleases (XhoI, SmaI, SacI and PvuII) and hybridization to heterologous chlDNA probes and to isolated melon chlDNA fragments. Four plastome-coded genes were located using heterologous probes. The overall organization and gene position of the melon plastome was found to be similar to that of tobacco and other angiosperm species. Restriction patterns based on digestion of the chlDNA with nine endonucleases were obtained in over 20 wild species and cultivated varieties of Cucumis. These led to mutational analysis of the restiction sites yielding the most parsimonious phylogenetic tree of the Cucumis plastome. Most African species from a compact group (Anguria group) which is distant from the melon, the cucumber and a few other species (C. sagittatus, C. metuliferus and C. humifructus). All of these are also far apart from each other. The distribution of polymorphic restriction sites along the Cucumis plastome is described and conservative regions as well as hot spots are suggested.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

Insertion polymorphism in pea chloroplast DNA

Summary The chloroplast DNA of higher plants is suitable for restriction endonuclease analysis due to its size and homogeneity. We have analysed 48 different cultivars of pea (Pisum sativum) with EcoRI and HindIII. Of these, only 24 show the standard genotype, the remaining 24 comprise four different classes of short insertions, three of which are found at the same site. Even though this kind of insertion polymorphism has not been detected elsewhere in the plant kingdom, it is consistent with the discovery that the chloroplast DNA of pea is destabilised through the loss of an inverted repeat.     

Electron microscopic localization of replication origins in Oenothera chloroplast DNA

Summary The origins of chloroplast DNA (cpDNA) replication were mapped in two plastome types of Oenothera in order to determine whether variation in the origin of cpDNA replication could account for the different transmission abilities associated with these plastomes. Two pairs of displacement loop (D-loop) initiation sites were observed on closed circular cpDNA molecules by electron microscopy. Each pair of D-loops was mapped to the inverted repeats of the Oenothera cpDNA by the analysis of restriction fragments. The starting points of the two adjacent D-loops are approximately 4 kb apart, bracketing the 16S rRNA gene. Although there are small DNA length variations near one of the D-loop initiation sites, no apparent differences in the number and the location of replication origins were observed between plastomes with the highest (type I) and lowest (type IV) transmission efficiencies.     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10⁻¹¹±1.4×10⁻¹¹ nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.