Plant and Cell Physiology (2005), Vol 46: 1462-1471  Pdf file Lössl et al. 2005 for Acrobat ReaderPDF file, 550 kb

Inducible Trans-Activation of Plastid Transgenes:
Expression of the R. eutropha phb Operon in Transplastomic Tobacco 

Andreas Lössl 1*, Karen Bohmert 2, Hans Harloff 3, Christian Eibl 4, Stefan Mühlbauer 4, and Hans-Ulrich Koop 5

1 Department of Applied Plant Sciences and Plant Biotechnology (DAPP), University of Natural Resources and Applied Life Sciences Vienna, Gregor-Mendel-Strasse 33, 1180 Vienna, Austria; Ludwig-Maximilians-Universität, Faculty of Biology, Department I, Botany, Menzinger Str. 67, D-80638 Munich, Germany
2 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany
3 Institut für Pflanzenbau und Pflanzenzüchtung, Christian-Albrechts-Universität Kiel, Olshausenstr. 40, 24098 Kiel, Germany
4 ICON Genetics AG, Research Centre Freising, Lise-Meitner-Str. 30, 85354 Freising, Germany
5 Ludwig-Maximilians-Universität, Faculty of Biology, Department I, Botany, Menzinger Str. 67, D-80638 Munich, Germany; ICON Genetics AG, Research Centre Freising, Lise-Meitner-Str. 30, 85354 Freising, Germany

* To whom correspondence should be addressed.
Andreas Lössl, E-mail: Ethanol inducibility


Abstract
Deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. Constraints like 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 up to 1383 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.

Keywords: Inducible transcription; Polyhydroxybutyrate; Plastid Transformation; Regulation; Renewable resources; Trans-activation system.

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Andreas Lössl, 2005