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.     

Plastid inheritance in Epilobium

Summary Interspecific hybrids of various Epilobium species have been produced in order to analyse plastid inheritance using restriction fragment polymorphisms of plastid DNA as markers. This analysis reveals that interspecific hybrids exhibit only the fragment pattern of the maternal plastome. Southern hybridization experiments using cloned species-specific plastid DNA fragments as markers confirm the maternal type of plastid inheritance in Epilobium, while providing at least a tenfold increase of sensitivity to detect restriction polymorphisms. Within the limit of detection even young seedlings contain no plastid DNA from the paternal parent. However, investigations of plastomes of large populations have provided evidence that a very low frequency of paternal plastid transmission can occur. Thus, the mechanism which ensures the elimination of paternal plastids is not 100% efficient. This suggestion is also supported by intraspecific reciprocal crosses between plants carrying mutant white and normal green plastids. While the offspring usually exhibit the maternal plastid type, a few cases indicate an apparent paternal plastid transmission.Abbreviations kbp kilobase pairs - ptDNA plastid DNA     

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.     

Cell type determines plastid transmission in tomato intergeneric somatic hybrids

Summary Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S+M, M+M and S+S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M+S and S+M fusion combinations. In contrast, for the M+M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S+M and M+S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M+M fusion, plastid and nucleotid input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.     

Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants

Different transgenic plants of Brassica napus, Brassica nigra, Datura innoxia and Vicia narbonensis expressing the hph gene under the control of the 35s promoter were co-cultivated with mycelial material of Aspergillus niger in microcosms under sterile conditions. A significantly higher number of hygromycin B-resistant colonies of re-isolated fungi was obtained if compared with co-cultures with non-transgenic plants. The hph gene and other foreign sequences could be detected in some of the resistant strains only for a short time after selection, indicating a rapid loss of foreign DNA. A more stable transgenic strain was obtained after co-culture with transgenic plants of D. innoxia including a high number of hph copies in their genome. DNA with detected pUC sequences was prepared to transform E. coli DH5. One of the recovered plasmids is shown to include pieces of the plant-transforming vector and a foreign sequence. The 35s-regulated expression of genes is studied in A. niger.     

Light and benzylaminopurine induce changes in ultrastructure and gene expression in plastids of Petunia hybrida cell cultures

Summary The regulatory effect of light and the cytokinin 6-benzylaminopurine (BA) on the plastid ultrastructure and plastid DNA gene expression is studied in white and mutant green cell suspension cultures of Petunia hybrida. By electron microscopy we show that both light and 6-benzylaminopurine induce the formation of thylakoid membranes and grana structures in plastids of the green cultures. For membrane formation in plastids of white cultures, light in combination with BA is required. Light and benzylaminopurine also influence the plastid DNA gene expression. By in-organello protein synthesis with isolated plastids we show that light as well as benzylaminopurine affects the synthesis of plastid DNA encoded proteins. A characteristic effect of benzylaminopurine on plastids from white and green cultures is the reduction in the synthesis of the CFI subunits of 55,000 and 57,000 D, and the reduction in the synthesis of large polypeptides with a molecular weight higher than 67,000 D. In contrast to benzylaminopurine, light only affects the DNA gene expression of plastids from white cell cultures, that are in a very early stage of plastid development. Light stimulates the synthesis of polypeptides with a molecular weight of 84,000, 70,000 and 46,000 D which are encoded by cpDNA in these white culture plastids. In green cell cultures both plastids with a etioplast-like phenotype and with a chloroplast like morphology synthesize similar polypeptides, resulting in the same polypeptide pattern. Our results indicate that qualitative differences in plastid DNA gene expression as an effect of light do occur but only in plastids at very early stages of chloroplast development. We observe a gradual reduction in the number of high molecular weight polypeptides at later stages of chloroplast development. This suggests that these large polypeptides are characteristic for plastids at an early developmental stage.Abbreviations LSU of RuBPCase large subunit of Ribulose-1, 5-bisphosphate carboxylase - CF₁ coupling factor of the ATPase complex - LCH chlorophyll a/b protein - BA 6-benzylaminopurine - cpDNA chloroplast DNA     

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.     

The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginfolia

This paper describes the analysis of chloroplast (cp) DNA and mitochondrial (mt) DNA in 21 somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia by means of Southern-blot hybridization. Each of these calli contained only one type of cpDNA; 14 had the N. plumbaginifolia (Np) type and seven the S. tuberosum (St) type. N. plumbaginifolia cpDNA was present in hybrids previously shown to contain predominantly N. plumbaginifolia chromosomes whereas hybrids in which S. tuberosum chromosomes predominated possessed cpDNA from potato. We have analyzed the mtDNA of these 21 somatic hybrid calli using four restriction enzyme/probe combinations. Most fusion products had only, or mostly, mtDNA fragments from the parent that predominated in the nucleus. The hybrids containing mtDNA fragments from only one parent (and new fragments) also possessed chloroplasts from the same species. The results suggest the existence of a strong nucleo-cytoplasmic incongruity which affects the genome composition of somatic hybrids between distantly related species.     

The <Emphasis Type="Italic">rpl5</Emphasis>–<Emphasis Type="Italic">rps14</Emphasis> mitochondrial region: a hot spot for DNA rearrangements in <Emphasis Type="Italic">Solanum</Emphasis> spp. somatic hybrids

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5–rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5–rps14 arrangements shown after somatic hybridization. Variability in the rpl5–rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.Contribution no. 39 from the Institute of Plant Genetics, Research Division of Portici.Communicated by A. Brennicke     

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.     

Biased organelle transmission in somatic hybrids ofLycopersicon esculentum andSolanum lycopersicoides

Summary Restriction fragment length polymorphisms (RFLPs) were used to determine the transmission of organelle genomes in somatic hybrid plants of tomato and its wild relativeSolanum lycopersicoides. Biased frequencies of organelle combinations were observed in a population of 70 somatic hybrid plants each derived from a separate callus. The plastids in 68 of 70 hybrids examined were fromL. esculentum. One of the remaining hybrids, plant 240, hadS. lycopersicoides plastids and the other, plant 63, had a mixture of parental plastids. Forty-six of the same 70 plants were analyzed for mtDNA and all had that ofS. lycopersicoides including plant 240. One of these hybrids had novel mtDNA fragments which mayhave resulted from recombination or rearrangement. The biased transmission may have resulted from an initial unequal input of organelles, differential replication of organelles, or nucleo-organelle incompatibility.Michigan Agricultural Experiment Station Journal Article No. 12538. Supported by Grant No. I-751-84R from BARD, The United States-Israel Binational Agricultural Research and Development Fund     

Plastid fusion as an agent to arrest sorting out

Summary Plastid fusions were noted in an ultrastrucutal study of a mutant of Hosta showing slow sorting out of plastid genes. These data suggest that fusions between wild type and mutant plastids might increase the mixing of plastid DNA and hence slow the process of sorting out. It is likely that peripheral reticula are involved in the process of plastid fusion between the mutant and wild-type plastids.     

Paternal plastid inheritance in alfalfa: plastid nucleoid number within generative cells correlates poorly with plastid number and male plastid transmission trength

Summary A previous study on alfalfa determined that the number of plastids/generative cell does not necessarily correlate with male plastid transmission strength in a given genotype. The objectives of the present study were to learn (1) whether plastid nucleoid number/generative cell is comparable to the number of plastids/generative cell, and (2) whether plastid nucleoid number/generative cell correlates with known male plastid transmission behavior in three alfalfa genotypes. Our results, which were based upon 150 generative cells examined by DAPI/epifluorescence microscopy, indicate that the mean plastid nucleoid number/generative cell is much less than the mean number of plastids/generative cell in genotype 7W (60 nucleoids/264 plastids) and genotype 301 (54 nucleoids/165 plastids). In genotype MS-5, mean plastid nucleoid number/generative cell (45) is similar to the mean number of plastids/generative cell (65). The significantly fewer plastid nucleoids/generative cell in MS-5, compared to that of 7W and 301, correlates positively with the relatively poor male plastid transmission strength of this genotype. However, the difference between the mean number of plastid nucleoids/generative cell in 7W and 301 is not significant, yet 301 is a much stronger transmitter of male plastids than is 7W.     

Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications

Summary The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.     

Lack of a functional plastid tRNACys gene is associated with loss of photosynthesis in a lineage of parasitic plants

Summary We recently reported that the gene for chloroplast tRNA^Cys(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parastic flowering plant in the family Orobanchaceae. Since this is the only tRNA^Cys gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosythetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA^Cys gene. Thus, the tRNA^Cys gene appears to have become non-functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only.     

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.     

Rhizosphere fungi of five plants in Egypt

75 species which belong to 25 genera were collected during this investigation. All of these fungi were previously isolated from Egyptian soils and other sources. The total count and the number of genera and species in the rhizosphere soil of the 5 plants were regularly higher than in non-rhizosphere soil. The composition of the rhizosphere fungus flora was considerably influenced by the type and the age of the plant. The most prevalent fungi in the rhizosphere of the test plants after 45, 90, and 150 days were as follows: in Helianthus annuus, Stachybotrys atra and Aspergillus niger; in Chrysanthemum coronarium, Cladosporium herbarum, A. sydowii and Penicillium funiculosum; in Nigella sativa, Fusarium moniliforme and A. sydowii; in Datura innoxia, A. fumigatus and A. terreus; in Hyoscyamus muticus, C. herbarum and A. sydowii; and in soil, P. funiculosum, A. terreus and A. niger. The counts of total fungi and most of the common fungal species, except in the case of Datura innoxia, reached their maxima after 90 days and sharply declined after 150 days.     

Expression of intron-encoded maturase-like polypeptides in potato chloroplasts

The trnK gene has been identified on a cloned plastid DNA fragment of potato (Solanum tuberosum cv Désirée). This gene codes for a tRNA-Lys and is interrupted by a 2.5-kb intron belonging to the group II organellar introns. In addition, this intervening sequence contains a long open reading frame potentially coding for a 509 amino-acid polypeptide (ORF509) related to mitochondrial intron-encoded maturases from fungi. The translational capacity of the trnK intron was first demonstrated in vitro in a prokaryotic DNA-directed expression system. In order to examine the expression of the intron in the potato plant, a synthetic peptide corresponding to the last nine amino acids of the predicted ORF509 product was used to raise antibodies. Westernblot experiments on chloroplast protein extracts, using a sensitive chemiluminescent detection system, identified polypeptides similar to in-vitro products. These results suggest that the trnK intron is expressed at the protein level in the plant. This is the first report of the in-vivo expression of an intron-encoded polypeptide in higher plant plastids.     

The peculiar distribution of class I and class II aldolases in diatoms and in red algae

Diatom plastids probably evolved by secondary endocytobiosis from a red alga that was up by a eukaryotic host cell. Apparently, this process increased the complexity of the intracellular distribution of metabolic enzymes. We identified genes encoding fructose-bisphosphate aldolases (FBA) in two centric (Odontella sinensis, Thalassiosira pseudonana) and one pennate (Phaeodactylum tricornutum) diatoms and found that four different aldolases are present in both groups: two plastid targeted class II enzymes (FBAC1 and FBAC2), one cytosolic class II (FBA3) and one cytosolic class I (FBA4) enzyme. The pennate Phaeodactylum possesses an additional plastidic class I enzyme (FBAC5). We verified the classification of the different aldolases in the diatoms by enzymatic characterization of isolated plastids and whole cell extracts. Interestingly, our results imply that in plastids of centric and pennate diatoms mainly either class I or class II aldolases are active. We also identified genes for both class I and class II aldolases in red algal EST databases, thus presenting a fascinating example of the reutilization and recompartmentalization of different aldolase isoenzymes during secondary endocytobiosis but as well demonstrating the limited use of metabolic enzymes as markers for the interpretation of phylogenetic histories in algae. The nucleotide sequences have been deposited at Genbank under the accession numbers AY116588, AY191866 and AY212505