Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins.

Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from purified P. hybrida mitochondria, specific initiation of DNA synthesis could only be observed starting within two fragments, oriA and oriB. When DNA synthesis incubations were performed with DNA templates consisting of both the A and B origins in the same plasmid in complementary strands, DNA synthesis first initiates in the A-origin, proceeds in the direction of the B-origin after which replication is also initiated in the B-origin. Based on these observations, a replication model for the P. hybrida mitochondrial genome is presented.

Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration.

Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 µl droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).

Transcriptional and post-transcriptional regulation of chloroplast gene expression in Petunia hybrida.

To study the control of differential gene expression during plastid biogenesis in Petunia hybrida, we have investigated the in vivo translation and transcription of the rbc L gene, coding for the large subunit of ribulose bisphosphate carboxylase (LSU), and the psa A gene, coding for P700 chlorophyll-a apoprotein (AP700). Differential expression of these plastid-encoded genes was studied in two developmentally different plastid systems, proplastid-like organelles from the green cell suspension AK2401 and mature chloroplasts from green leaves. In vivo translation of rbc L and psa A transcripts was analysed using specific antibodies. Specific transcript levels were analysed using internal fragments of the rbc L and psa A genes. A standardization procedure was used so that a direct correlation could be made between the amount of products and gene copy number. In Petunia hybrida the amount of LSU polypeptides present in both plastid types does not correspond to the amount of specific mRNA for the gene. Although the rbc L transcripts are present in both plastid types, the LSU protein is only present in green leaf plastids and not in cell culture plastids. In vitro translation of isolated rbc L transcripts give similar results, thereby suggesting that differences in the primary structure of the transcripts are responsible for the observed discrepancy. In contrast to this, the amount of AP700 polypeptides does correspond to the amount of the psa A transcripts. Therefore, our results indicate that the expression of chloroplast genes during plastid biogenesis takes place on at least two different levels: expression of the rbc L gene is regulated post-transcriptionally while expression of the psa A gene is regulated at the transcriptional level.

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida.

Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm(-3) in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2-30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).

Physical mapping, nucleotide sequencing and expression in E. coli minicells of the gene for the large subunit of ribulose bisphosphate carboxylase from Petunia hybrida.

The Petunia hybrida rbcL gene was identified and located on the physical map within the Sall S9 fragment of the Petunia hybrida cpDNA by heterologous hybridization with the cloned rbcL gene of spinach (pSoc3BE148). In E. coli minicells harbouring the S9 fragment inserted into pBR322, the rbcL polypeptide is synthesized as was shown by molecular weight determination, immunoprecipitation and proteolytic digestion. However, the size of the rbcL polypeptide synthesized in minicells appeared to be dependent on the orientation of the S9 fragment in pBR322. In minicells harbouring the S9 fragment inserted into pBR322 in the clockwise orientation the molecular weight of the rbcL polypeptide is approximately 53 kD, whereas in minicells harbouring the S9 fragment in the opposite orientation, the rbcL polypeptide synthesized has a molecular weight of 52 kD. The difference in molecular weight of the two rbcL polypeptides is the result of transcription and translation into the flanking pBR322 sequences. This is due to the absence of the terminal part (6 codons), including the translation stop codon, of the rbcL gene on the cloned S9 fragment as was determined by nucleotide sequencing. The observed expression of the cloned part of the rbcL gene of Petunia hybrida indicates that the E. coli minicell system can be used as a suitable and convenient system for the identification and physical mapping of chloroplast genes.Comparison of the sequence of the untranslated 3'-end of the rbcL gene of Petunia hybrida with that of Nicotiana tabacum revealed a striking similarity of the region in which stem and loop structures can be formed that are most likely involved in termination of transcription of the rbcL gene. This region appears to be highly conserved in the rbcL genes of P. hybrida, N. tabacum, S. oleracea and Z. mays.

Physical mapping of genes for chloroplast DNA encoded subunit polypeptides of the ATPsynthase complex from Petunia hybrida.

Escherichia coli minicells harbouring the cloned restriction fragment Sall S9 from P. hybrida chloroplast DNA synthesize the beta and epsilon polypeptide subunits of the CF1 component of the chloroplast ATPsynthase complex. The polypeptides were identified by molecular weight determination and immunoprecipitation. The position of the atpB and the atpE gene, encoding respectively the beta and epsilon subunit, on the Sall S9 fragment was determined in more detail by studying polypeptide synthesis directed by subclones of the S9 fragment in E. coli minicells. The atpB and atpE genes are located close to the rbcL gene, the distance between the rbcL gene and atpB gene being approximately 770 bp. Analysis of the expression of subclones of the S9 fragment in E. coli minicells also revealed that the atpE gene can be transcribed and translated independently of the expression of the atpB gene.The location of the genes coding for the alpha subunit (atpA gene) and the proteolipid subunit III of CF0 (atpH) of the ATPsynthase complex on the physical map of P. hybrida cpDNA was determined by hybridization of restriction enzyme digests of petunia cpDNA with cloned cpDNA fragments from Spirodela and wheat, containing internal parts of respectively the atpA and the atpH gene. The two genes map close together within a region of 5.2 kbp on the physical map of P. hybrida cpDNA. The distance between the atpA gene and the atpB and atpE genes is approximately 42 kbp.

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast.

Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0-28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.

Plastid gene expression in a yellow-green leaf mutant of Petunia hybrida.

We have analyzed the morphology and gene expression in plastids of a yellow-green leaf mutant of Petunia hybrida (E 5059). Under normal light intensities (20,000 lx), yellow-green leaves develop with a typical proplastid morphology (few membranes, incomplete stacking). When such plants are grown under low light intensities (3,000 lx), the newly formed leaves are green. The plastids in these green leaves have a wild-type like chloroplast morphology (thylakoids and grana structure). Pre-existing green leaves remain green in 20,000 lx, indicating that chlorophyll is not degraded. An analysis of polypeptides synthesized in isolated plastids from the yellow-green and green leaves of this mutant plant shows several differences. In the yellow-green leaf plastids only a very small amount of the large subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase) is present, while in green plastids this polypeptide is present in much higher amounts. Hybridization experiments indicated that in plastids from the yellow-green leaves the mRNA coding for the large subunit polypeptide is present in much lower amounts than in plastids from the 3,000 lx green leaves of this mutant or in chloroplasts from wild type plants. These results indicate regulation at the mRNA level. Furthermore, in yellow-green leaf plastids eleven polypeptides are present with high molecular wieght (higher than 67,000 d). Five of them are synthesized by the yellow-green leaf plastid itself. Such high molecular weight polypeptides are also synthesized by proplastids isolated from white petunia cell suspension cultures, but are not synthesized by 3,000 lx green leaf plastids, or by isolated normal leaf chloroplasts. These results indicate that the synthesis of these polypeptides is specific for the proplastid stage of chloroplast biogenesis.

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

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.

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia.

Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

Protein synthesis in Petunia hybrida chloroplasts isolated from leaves and cell cultures.

Isolation and incubation conditions were established for Petunia hybrida chloroplasts capable of performing in vitro protein and RNA synthesis. Under these conditions, chloroplasts from leaves as well as from the non-photoautotrophic mutant green cell culture AK-2401 are able to incorporate labeled amino acids into polypeptides. Intact chloroplasts can use light as an energy source; photosynthetically-inactive chloroplasts require the addition for ATP for this protein synthesis. Sodium dodecylsulphate polyacrylamide slab gel electrophoresis shows that in isolated leaf chloroplasts at least twenty-five radioactive polypeptide species are synthesized. The three major products synthesized have molecular weights of 52,000, 32,000 and 17,000. Coomassie brilliant-bluestained polypeptide patterns from plastids isolated from the mutant green cell culture AK-2401 differ considerably from those obtained from leaf chloroplasts. The pattern of radioactive polypeptides synthesized in these isolated cell culture plastids also shows differences. These results indicate that the difference in developmental stage observed between plastids from the cell culture AK-2401 and leaves is reflected in an altered expression of the chloroplast DNA.

Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA.

A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida. The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons. This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA. Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3. This corresponds with a GC-content of 39.3 +/- 0.5%. A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I. The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation. The inverted repeat regions have a minimum length of 10 x 10(6) daltons. Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs.

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures.

By using mercury(II)-chloride (HgCl2) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N'-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl2 and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10(-5) and 2.0 x 10(-6) for HgCl2 and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5-40 µg/ml) that had no observable effect on the survival of the mutagenized cultures.

Bacteriocinogenic Clo DF13 minicells of Escherichia coli synthesize a protein that accounts for immunity to bacteriocin Clo DF13: purification and characterization of the immunity protein.

Cloacin DF13 inhibits protein synthesis both in vivo and in vitro by inactivation of ribosomes. In this paper we describe the purification, from purified bacteriocinogenic plasmid DF13 (Clo DF13)-harboring minicells of Escherichia coli, of an acidic protein (molecular weight about 10,000) that prevents the inhibition of in vitro protein synthesis by cloacin DF13. It turned out that this protein is one of three Clo DF13 plasmid-specific polypeptides that are synthesized by the noninduced Clo DF13 plasmid in minicells. Furthermore, we observed that, after induction with mitomycin C, the amount of immunity protein is only slightly increased, whereas the cloacin DF13 synthesis is increased about 40 times. These results imply that the genes that code for immunity protein and cloacin DF13 are not located in the same regulatory unit. Under noninduced conditions, apparently an excess of immunity protein is synthesized, because this amount of immunity protein is even sufficient to neutralize the strongly increased amount of cloacin after induction, since growth of induced cloacinogenic cells is continued.

Bacteriocinogenic Clo DF13 minicells of Escherichia coli synthesize a protein that accounts for immunity to bacteriocin Clo DF16: action of the immunity protein in vivo and in vitro.

The Clo DF13 plasmid-specific immunity protein is able to prevent the inhibitory effect of cloacin DF13 on in vitro protein synthesis. We have shown, by gel filtration, that direct binding of the Clo DF13 immunity protein to cloacin occurs in vitro. This cloacin DF13-immunity protein complex is rather stable, and the cloacin present in the complex is no longer able to cause inhibition of in vitro protein synthesis. The binding of immunity protein to cloacin DF13 is rather specific because the Clo DF13 immunity protein does not bind to in vitro inactive cloacin and binds very poorly to the closely related bacteriocin colicin E3. Furthermore, we present data which strongly suggest that in vitro at least a fourfold excess of immunity protein is required to ensure that every cloacin molecule is inactivated by cloacin-immunity protein complex formation. Only a fraction (an about equimolar amount) of the immunity protein molecules, however, actually binds to cloacin DF13. The existence of an immunity protein-cloacin complex in vivo was concluded from the observation that cloacin, purified by chromatography on diethyl-(2-hydroxypropyl)-aminoethyl Sephadex in the absence of urea, contains an about equimolar amount of a second protein which comigrates with immunity protein on sodium dodecyl sulfate-polyacrylamide and urea-polyacrylamide gels. In an in vitro protein-synthesizing system, this component appeared to behave identical to the Clo DF13 immunity protein. The purified immunity protein-containing cloacin was at least 80 times less active in inhibiting in vitro protein synthesis, compared to cloacin, free of immunity protein. These data imply that few, if any, cloacin DF13 molecules are present in cloacinogenic cells as active, free cloacin molecules.

Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid Clo DF13.

After nitrosoguanidine mutagenesis, strain Escherichia coli P678-54, bacteriocinogenic for Clo DF13, yielded a mutant strain that showed an enhanced bacteriocin production. The results from conjugation experiments indicated that the mutation, responsible for the enhanced bacteriocin production, is located on the Clo DF13 plasmid. The following properties of strains harboring the mutant Clo DF13 plasmid could be observed. (i) The bacteriocin production in these strains can be further enhanced at least fourfold by mitomycin C. (ii) The fraction of spontaneously induced cells, as revealed by lacunae experiments, in cultures of these strains is about nine times higher than in cultures of wild-type Clo DF13-harboring strains. (iii) Chromosomeless minicells from strain P678-54 harboring the mutant Clo DF13 plasmid synthesize about six times more deoxyribonucleic acid, ribonucleic acid, and protein as compared to wild-type Clo DF13-harboring minicells. (iv) Analysis of this mutant Clo DF13-specific ribonucleic acid and protein on polyacrylamide gels revealed mainly the same ribonucleic acid and polypeptide species as synthesized by the wild-type Clo DF13 minicells, but in larger amounts (Kool et al., 1974). (v) Segregation experiments, using a strain with temperature-sensitive polymerase I, show that mutant Clo DF13-harboring cells contain an average of 70 Clo DF13 copies per cell, whereas wild-type Clo DF13-harboring cells contain only about 10 Clo DF13 copies per cell. The data presented in this paper indicate that the mutation on the Clo DF13 plasmid leads to an altered control of Clo DF13 replication and results in an enhanced number of Clo DF13 copies per cell. As a secondary effect, this enhanced number of Clo DF13 copies enhances the probability of "spontaneous" induction per cell. Since the mutation is plasmid specific and affects the number of plasmid copies produced, one can conclude that the Clo DF13 plasmid is not dependent solely on chromosomal information, but that at least plasmid base sequences are involved in Clo DF13 plasmid replication.

Identification of messenger ribonucleic acids and proteins synthesized by the bacteriocinogenic factor Clo DF13 in purified minicells of Escherichia coli.

It has previously been shown that the cloacinogenic factor Clo DF13 (Clo DF13) segregates into minicells of strain Escherichia coli P678-54 that harbors Clo DF13 and that this Clo DF13 factor is the only deoxyribonucleic acid (DNA) present in these otherwise chromosomeless minicells. The study reported here shows that minicells prepared from P678-54(Clo DF13) are able to incorporate radioactive precursors into ribonucleic acid (RNA) and protein. The RNA synthesized in these purified minicells is Clo DF13 specific, as shown by RNA-DNA hybridization experiments. The results indicate that all the de novo synthesized gene products in Clo DF13 minicells are Clo DF13 specific. Polyacrylamide gel electrophoretic patterns show that in these minicells at least three polypeptides (molecular weight about 70,000, 20,000, and 11,000) and one major species of messenger RNA (mRNA) (S value about 21.3) are synthesized. To investigate the factor in its induced state, we isolated a Clo DF13 mutant with an enhanced level of cloacin production. Minicells harboring this Clo DF13 mutant produce five additional polypeptides (molecular weight about 58,000, 44,000, 28,000, 16,000, and 14,000). Three additional mRNA species (S value about 19.5, 14, and 12) could be distinguished. The total molecular weight of the eight polypeptides corresponds to 85% of the total coding capacity of the mRNAs (303,000). The total molecular weight of the four mRNAs is 2.55 x 10(6), which covers 85% of the Clo DF13 DNA (molecular weight 6 x 10(6)).