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     

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.     

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     

Genome differentiation by GISH in interspecific and intergeneric hybrids of tomato and related nightshades

We employed genomic in situ hybridization to analyze the chromosomal constitution and pairing of interspecific and intergeneric hybrids involving cultivated tomato (Lycopersicon esculentum) and two related wild nightshade species, Solanum lycopersicoides and S. sitiens. Using standard stringency conditions, the tomato genome was readily distinguished from that of the two nightshades, whereas the latter were only distinguishable under increased stringency. These observations indicate a more distant phylogenetic relationship between L. esculentum and the Solanum group, and suggest S. lycopersicoides and S. sitiens share a high degree of sequence homology. Chromosomal associations during meiosis of interspecific and intergeneric hybrids were consistent with these relationships: chromosomes of F₁ L. esculentum×S. lycopersicoides and F₁ L. esculentum×S. sitiens hybrids frequently formed univalents during diakinesis. In contrast, F₁ S. lycopersicoides×S. sitiens hybrids showed complete bivalent formation. L. esculentum×S. sitiens hybrids, including the F₁ plants, a monosomic addition, and an allotetraploid, showed lower frequencies of pairing between homeologous chromosomes than the corresponding L. esculentum×S. lycopersicoides genotypes. A trigenomic 2n+14 hybrid, with 12 extra chromosomes from S. sitiens and 2 from S. lycopers icoides, displayed mostly homologous chromosome associations. The distribution of rDNA genes appeared similar in the three genomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Molecular analysis of mitochondrial DNA from tomato cell suspension cultures

Summary Mitochondrial DNA (mtDNA) from Lycopersicon esculentum was purified from cell suspension cultures. The DNA, isolated from mitochondria purified by two successive sucrose density gradients, was uncontaminated with nuclear DNA or DNA from proplastids. The total molecular weights of BamHI, BglI, and BglII fragments indicate a mitochondrial genome size of at least 270 kb. Cross hybridization between tomato mtDNA and cloned spinach plastid genes revealed some homology. In hybridization experiments using cloned mitochondrial rRNA genes and BamHI digested total mtDNA the presence of recombination repeats is demonstrated.     

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.     

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.     

Infection of cowpea mesophyll protoplasts with cowpea mosaic virus.

Mesophyll protoplasts were isolated from the primary leaves of cowpeas by a one-step procedure using a mixture of Macerozyme and Cellulase. The protoplasts were inoculated with cowpea mosaic virus and virus multiplication was shown to occur by measuring the virus infectivity at various times after inoculation. Eighty to ninety-six percent of the protoplasts were infected as shown by fluorescent antibody staining. More than 10⁸ progeny virus particles were produced per infected protoplast. A cytopathic structure similar to the structure found in CPMV infected leaf cells occurred in infected protoplasts. Poly-l-ornithine was not essential for CPMV infection of the cowpea mesophyll protoplasts, but it had a stimulating effect. This is the first protoplast system after the tobacco mesophyll protoplast system suitable for virus infection studies. As cowpeas belong to a plant family other than tobacco, the new protoplasts allow comparative infection studies with plant viruses.     

Loss of mt +-derived zygotic chloropiast DNA is associated with a lethal allele in Chlamydomonas monoica

Summary The Chlamydomonas monoica mutant allele mtl-1, is associated with the formation of nonviable zygospores following self-mating of the mutant strain. Furthermore, mtl-1 heterozygote populations show a 50% reduction in germination frequency and no transmission of a chloroplast antibiotic resistance marker carried by the mtl-1 parent. To determine whether the effects on zygospore viability and chloroplast gene transmission resulted from the direct involvement of the mtl-1 locus in the control of mt ⁺-directed uniparental inheritance of chloroplast DNA (cpDNA), we have used the DNA-specific fluorochrome DAPI to follow the fate of cpDNA during the maturation of zygotes. Throughout the first few hours after the initial fusion of gametes, the young zygotes show DAPI-fluorescent nucleoids distributed symmetrically around the region of nuclear fusion, and presumably located within both of the parental chloroplasts. Wild-type and mtl-1 mutant zygotes show similar early staining patterns. As the zygotes age, the staining patterns become asymmetric for the wildtype population, with all of the visible cytoplasmic nucleoids restricted to one side of the zygote. In contrast, mtl-1 homozygotes appear to lose cytoplasmic nucleoids from both sides of the zygote simultaneously and within 24 h are apparently devoid of cpDNA. By introducing a mutation which arrests cell fusion (and prevents plastid fusion), we can show that (1) the asymmetric nucleoid distribution in wildtype zygotes results from the loss of nucleoids from one gamete in each mating event, and (2) the additional loss of cpDNA in mtl-1 homozygotes does not require contact between parental plastids (thus the nuclease responsible for cpDNA degradation is not sequestered within the chloroplast of one gamete). We propose that the mtl-1 mutant strain is defective for a process which normally protects cpDNA of mt ⁺ origin.     

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     

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.     

Investigations on the infection of cucumber mesophyll protoplasts with cucumber mosaic virus

Summary Isolated protoplasts from the first leaf mesophyll of cucumber plants have been successfully infectedin vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowpea; however, viral RNA extraction improved the bioassay technique. Attempts to optimizo inoculation and incubation of protoplasts are outlined, incorporating the improved assay.     

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.     

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     

Plastid genomes of the Rhodophyta and Chromophyta constitute a distinct lineage which differs from that of the Chlorophyta and have a composite phylogenetic origin,perhaps like that of the Euglenophyta

Summary A phylogenetic tree has been constructed from comparisons of entire 16S rRNA gene sequences from different prokaryotes and from several algal plastids. According to this study, and to previous work on the ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) large and small subunit genes, we postulate that: (1) rhodophyte and chromophyte plastid genomes have a common, composite phylogenetic origin which implies at least two different ancestors, a cyanobacterial and a -proteobacterial ancestor; (2) chlorophyte (green algae and land plants) plastids have a cyanobacterial ancestor which probably differs from that of rhodophyte and chromophyte plastids, and in any case constitute a different lineage; (3) euglenophyte plastid genomes also seem to have a composite phylogenetic origin which involves two different lineages.     

Preferential amplification of mitochondrial DNA fragments in somatic hybrids of the Gramineae

Summary Somatic hybrid cell lines of Pennisetum americanum + Panicum maximum, and of Pennisetum americanum + Saccharum officinarum display unique mitochondrial DNA (mtDNA) restriction patterns suggestive of mitochondrial fusion and recombination. Apparent recombinant fragments of the hybrids were recovered, cloned, and hybridized to parental and somatic hybrid mtDNAs. In each somatic hybrid, novel fragments were found to be present at low copy number in one or both of the parental mtDNAs, and amplified 15–30 times in the hybrids. In pearl millet-sugarcane somatic hybrid cells, the amplification does not appear to involve enhanced recombination. The presumably amplified restriction fragment of the pearl millet-Guinea grass somatic hybrids is a junction fragment of a repeat, present in low copy number in both parents, and in high copy number in the hybrids. Thus protoplast and probable mitochondrial fusion results in a marked shift in the direction of mtDNA recombination events. We conclude that amplification of parental mtDNA fragments is a common event in somatic hybrid cells of these Gramineae.