Plastid inheritance in Pisum sativum L.

Summary Cultivar variability for levels of plastid DNA (cpDNA) in the germ cell line of germinated pea pollen has suggested the possibility of biparental plastid transmission. In order to examine this possibility further, RFLP markers were used to follow the transmission of cpDNA from parents to their F₁ offspring. Results from these inheritance studies clearly indicate the presence of only maternal plastid markers in the F₁ progeny of each cross examined, irrespective of the pollen cpDNA levels of the paternal parent. The same result is obtained for F₁ progeny produced from crosses using pollen characterized by comparatively high cpDNA content, even when offspring are sampled at early developmental stages. Thus, there appears to be little correspondence between pollen cytological data indicating potential paternal plastid transmission and data from molecular marker studies confirming that P. sativum generally follows a uni-parental-maternal mode of plastid inheritance. Insufficient F₁ progeny were examined to exclude instances of trace biparentalism.     

Plastid inheritance in Oenothera: paternal input may influence transmission patterns

Summary Prior genetic analysis of Oenothera to assess the mechanism(s) controlling differential (biparental) plastid transmission patterns have indicated that the plastome plays an integral role. However, the influence of putative variation in paternal plastid input remains unclear. Pollen collected from Oenothera hookeri plants containing one of four different plastome types (I–IV) in a constant nuclear background (A₁A₁) was examined cytologically by DAPI/ epifluorescence microscopy. The number of plastid DNA aggregates per pollen generative cell was found to differ significantly. Plants containing plastome types I or II displayed an average of about ten plastid DNA aggregates per generative cell whereas plants containing types III or IV displayed, on average, 15 plastid DNA aggregates. The potential paternal plastid contribution to the egg cell at syngamy (III=IV>I=II) differs from the previously determined survival frequencies of the same four plastid types (I>III>II>IV) progeny.Scientific article no. A-5036, contribution no. 8084 of the Maryland Agricultural Experimental Station     

Cultivar variability for the presence of plastid DNA in pollen of Pisum sativum L.: implications for plastid transmission

Summary The mode of plastid transmission in the garden pea (Pisum sativum L.) was analyzed cytologically using the DNA-fluorochrome 4,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. The reproductive cells of mature pollen obtained from 12 inbred lines and cv Early Alaska were examined for the presence or absence of DAPI-stained plastid DNA aggregates. Plastid DNA was detected in all 13 pea lines examined, although there was variability with regard to the percentage of pollen graines showing plastid DNA aggregates of generative cells (ranging from 3% in accession 82-12r to 65% in accession 82-14n). These cytological results may indicate genetic variability for plastic DNA inheritance in the garden pea. This paper is dedicated to the memory of Gerald A. Marx     

Control of organelle transmission in Chlorophytum

Summary In a study of pollen ultrastructure in Chlorophytum comosum plastids were found to be present and apparently unaltered in both vegetative and generative cells. These ultrastructural data are consistent with the biparental transmission of plastids in this genus. However, mitochondria appear degenerate when compared to leaf mitochondria, exhibiting small myelin-like figures in addition to an abnormal cristae system. Since the plastids are not degenerate in the pollen of this species it appears that the transmission genetics of the mitochondria are determined separately from that of the plastid in higher plants.     

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

Summary The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.     

Differentiation of species and strains among filamentous fungi by DNA fingerprinting

Summary We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)₈, (GTG)₅ and (GACA)₄, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)₄ produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.     

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD)

Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonalty based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

rps10, unreported for plastid DNAs,is located on the cyanelle genome of Cyanophora paradoxa and is contranscribed with the str operon genes

Summary rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the S10 operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.     

Structure and expression of cytochrome f in an Oenothera plastome mutant

Summary The chloroplast mutant pm7 is one of a number of mutants derived from the plastome mutator (pm) line of Oenothera hookeri, strain Johansen. Immunoblotting showed that this mutant accumulates a protein that is cross-antigenic with cytochrome f, but five kilodaltons larger than the mature wild-type protein. Since cytochrome f is known to be translated on plastid ribosomes as a precursor with an amino-terminal extension, it is proposed that the unprocessed cytochrome f precursor accumulates in pm7. In addition to this precursor-sized cytochrome f protein, some mature-sized cytochrome f was also found in the mutant plastids. The pm7 mutation is inherited in a non-Mendelian fashion; but no alterations in chloroplast DNA restriction patterns, or differences in DNA sequence in the region encoding cytochrome f, were found in a comparison of the wild-type and pm7 chloroplast DNAs. Although the mutant was capable of synthesizing heme, no covalently-bound heme, normally found associated with mature, functional, cytochrome f was detected in the mutant at sizes expected for the presumed precursor, or for mature cytochrome f. These results indicate that the aberrant accumulation of a precursor-sized cytochrome f in pm7 is not due to a lesion directly in the plastid gene encoding cytochrome f, petA, or to a deficiency in the ability of the mutant plastids to synthesize or accumulate heme.     

Chloroplast DNA diversity within and among populations of Trifolium pratense

Summary In contrast to animal mitochondrial DNA, intraspecific variation in chloroplast DNA is thought to be very rare. This presumption has prevented the application to plant population biology of the diversity of molecular genetic techniques now well established for animal mitochondrial DNA. In Trifolium pratense, however, extensive intrapopulational variation does exist. In two paper I report a characterization of unprecedented restriction fragment profile variation within single populations. Populations typically contain a common genotype and many rare ones; often the rare genotypes differ from population to population. While both nucleon and nucleotide diversity, as well as estimates of Wright's F _ST, indicate a large within-population component and relatively little diversity among populations, the distribution of plastid genotype frequencies in each population is not homogeneous. Estimates of migration rate based on chloroplast DNA genotypes suggest a moderate number of migrants per generation. The unusually high level of genetic variation in Trifolium chloroplast DNA provides the first opportunity to use the plastid genome of plants to study population differentiation. Furthermore, it suggests that the plastid genome may not be as invariant as previously believed, but may instead exhibit high levels of genetic diversity at the population level.     

Non-random plastid segregation in somatic hybrids of Datura innoxia with several other Solanaceous species

Summary A non-random plastid segregation was found in somatic hybrids of Datura innoxia with seven different Solanaceous species. 14 out of 17 examined somatic hybrids showed the plastid features of Datura innoxia. Within the limits of sensitivity of the applied methods, one line could be shown to contain mixed plastids. Since sexual offspring of this line contains only one set of plastids, it is assumed that this is probably a periclinal chimaera due to the plastome, i.e., the plastid mixture is present on a plant rather than a cell level.     

Phylogenetic implication of heterogeneity of the nontranscribed spacer of rDNA repeating unit in various Neurospora and related fungal species

Summary Nontranscribed spacer (NTS) regions of ribosomal (r)RNA genes are non-conserved and are shown to be useful for phylogenetic studies. ³²P-labelled N. crassa NTS pCC3400 DNA, was used as a molecular probe to hybridize Southern blots of genomic DNAs obtained from Neurospora, Gelacinospora, Sordaria, bacteria, plants, and animals. Our studies conclude that: (a) the homotahllic species of Neurospora should not belong to genus Gelacinospora (a historical question) and that Neurospora homothallic species are closer to Gelacinospora than to Sordaria; and that (b) all of the filamentous fungal species tested are indeed closer to the higher plant genome than to higher primate animal genome based on shared restriction sites of 12 enzymes. Our studies also demonstrate the usefulness of nontranscribed rRNA gene probes in resolving questions regarding phylogenetic relatedness between widely separated organisms using the parsimony principle based on mutation sites from DNA restriction maps; it has not been possible to do this using DNA: DNA hybridization procedures that involved the total genome.     

Location and nucleotide sequence of the pre-apocytochrome f gene on the Oenothera hookeri plastid chromosome (Euoenothera plastome I)

Summary The gene for pre-apocytochrome f has been mapped by blot hybridization on a 2.4 kbp HindIII fragment of the circular plastid chromosome of Oenothera hookeii employing probes from the corresponding spinach gene. The gene is located distal to the gene for the ATP synthase subunit alpha, at the border of the 45 kbp inversion that distinguishes spinach and Oenothera plastid chromosomes. Both genes are transcribed in the same direction. Nucleotide sequence analysis reveals a single open reading frame encoding 318 amino acids of which 285 comprise the mature polypeptide and another 33 residues represent probably a N-terminal signal sequence. The putative pre-sequence is 2 residues shorter than those known from the spinach, wheat and pea protein. The deduced amino acid sequences of f cytochromes from the four plant species show over 80% conservation, maintaining the structural characteristics of the protein.     

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

Summary A 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.     

Investigation of plant organellar DNAs by pulsed-field gel electrophoresis

Mitochondrial (mt) DNAs from several higher-plant species (Arabidopsis thaliana, Beta vulgaris, Brassica hirta, Chenopodium album, Oenothera berteriana, Zea mays) were separated by pulsed-field gel electrophoresis (PFGE). Hybridization of the separated DNA with mtDNA-specific probes revealed an identical distribution of mtDNA sequences in all cases: part of the DNA formed a smear of linear molecules migrating into the gel, the rest remained in the well. Hybridization signals in the compression zone of the gels disappeared after RNase or alkaline treatment. It was shown that the linear molecules are not products of unspecific degradation by nucleases. All plastid (pt) DNA from leaves of Nicotiana tabacum remained in the well after PFGE. Separation of linear monomers and oligomers of the chloroplast chromosomes of N. tabacum was achieved by mild DNase treatment of the well-bound DNA. DNase treatment of well-bound mtDNA, however, generated a smear of linear molecules. PtDNA from cultured cells of C. album was found after PFGE to be partly well-bound, and partly separated into linear molecules with sizes of monomeric and oligomeric chromosomes. The ease with which it was possible to detect large linear molecules of plastid DNA indicates that shearing forces alone can not explain the smear of linear molecules obtained after PFGE of mtDNA. The results are discussed in relation to the structural organization of the mt genome of higher plants.     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).     

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones

Summary Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Extraordinary stability of IgE-binding Parietaria pollen allergens in relation to chemically bound flavonoids

It is known that the skin-active and IgE-binding components in Parietaria pollen extracts are not restricted to the predominant protein allergens of M_r 12 000–15 000, but are present as well among the naturally occurring constituents of M_r < 10 000. Indeed, the IgE-binding Parietaria pollen components are quite heterogeneous, ranging from high- to low-molecular mass, whereby the IgE-binding epitopes display an unusual chemical stability. Furthermore, the pollen of Parietaria species demonstrably contain a high proportion of flavonoid pigments. Since these pollen grains cannot be collected entirely free from non-pollen plant parts, the usual allergenic extracts of Parietaria encompass both the polyphenolic substrate molecules and the enzyme polyphenoloxidase as ingredients for the oxidative generation of flavonol-protein conjugates during the extraction process. In the present work this is illustrated by spectroscopic analyses of the free and bound flavonoids in Parietaria pollen extracts, as well as of the peptide fragments produced from the allergenic proteins by enzymatic or chemical hydrolysis. None of these relatively harsh treatments had a significant effect on the IgE-binding properties of the allergenic (sub-)components, even though detectable proteins in isoelectric focusing and immunoblotting were lost. It is proposed that the extraordinary stability of IgE-binding Parietaria components over a wide molecular range may be attributed to chromophoric flavonoid side-chains as (parts of) the corresponding B-cell epitopes.