Inducible trans-activation of plastid transgenes: expression of the R. eutropha phb operon in transplastomic tobacco.

Deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. Constraints such as growth inhibition and male sterility have been observed in plastid transformants containing the phb operon encoding the genes required for the production of the polyester polyhydroxybutyric acid (PHB). In order to induce PHB synthesis in tobacco in a well-timed manner, we have constructed a trans-activation system to regulate transcription of the phb operon in plastids. This system consists of a nuclear-located, ethanol-inducible T7RNA polymerase (T7RNAP) which is targeted to plastids harboring the phb operon under control of T7 regulatory elements. Following treatment with 5% ethanol, moderate induction of PHB synthesis was found. PHB amounts reached 1,383 ppm in dry weight, and an overall background activity of 171 ppm was measured in uninduced tissues. On the transcriptional level, T7RNAP induction was proven and we found that the phb operon is transcribed into at least two mRNAs. Without ethanol induction, development of flowers and fertile seeds was possible. Thus, the main problem of inhibitory transgene expression was solved. Our results show that this inducible trans-activation system could serve as an alternative to constitutive expression of transgenes in the plastome.

Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis.

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.