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.

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.

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.

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.