Inducible Trans-activation of Plastid Transgenes: Expression of the R. eutrophaphb Operon in Transplastomic Tobacco

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Abstract
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.