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.

Upon transfer of lysed chloroplasts from darkness to light, the accumulation of membrane and stromal chloroplast proteins is strictly regulated at the level of translation elongation. In darkness, translation elongation is retarded even in the presence of exogenously added ATP and dithiothreitol. In the light, addition of the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibits translation elongation even in the presence of ATP. This inhibition can be overcome by addition of artificial electron donors in the presence of light, but not in darkness. Electron flow between photosystem II and I induced by far red light of 730 nm is sufficient for the activation of translation elongation. This activation can also be obtained by electron donors to photosystem I, which transport protons into the thylakoid lumen. Release of the proton gradient by uncouplers prevents the light-dependent activation of translation elongation. Also, the induction of translation activation is switched off rapidly upon transfer from light to darkness. Hence, we propose that the formation of a photosynthetic proton gradient across the thylakoid membrane activates translation elongation in chloroplasts.Protein expression in plastids of algae and higher plants is regulated by light during the light-dependent development of chloroplasts as well as during the light-dark transitions of fully differentiated chloroplasts. A great proportion of the regulatory mechanisms were found to be mediated at a posttranscriptional level, as the mRNA levels of many of the concerned genes were little or not affected (1-4). Most of the posttranscriptional changes of protein expression were explained by a regulation of translation initiation; the 5Ј-untranslated region of the psbA gene encoding the reaction center protein D1 was shown to direct light-dependent accumulation of a reporter gene in transformed tobacco plants (5), and proteins binding to the 5Ј-regions of several chloroplast genes were observed in Chlamydomonas reinhardtii (6). Light-dependent binding of proteins to the 5Ј-untranslated region of the psbA gene was shown to be responsible for the light-dependent translation initiation in Chlamydomonas (7) and in spinach (8).Other examples of light-regulated protein expression were explained by translation elongation control; the mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase was not translated in the dark but remained bound to polysomes in Amaranthus (9). Accumulation of D1 translation intermediates in the dark was observed in pea chloroplasts (10, 11) and in spinach (12).Different explanations were given of how light regulated translation. Several groups attributed reduced protein expression in the dark to the reduced level of ATP in the chloroplast in the absence of photosynthetic activity (13,14). Conversely, reduced translation initiation of the psbA gene has been explained by the increased level of ADP in the dark (15). Binding of an initiation complex to the psbA mRNA was proposed to be regulated by ...

SummarySequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild-type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.

Intact and lysed chloroplasts isolated from the day or night phase of seedling growth exhibit a higher rate of [ 35 S]Met incorporation into the D1 protein in the light than in darkness. In the presence of the translation initiation inhibitor lincomycin, radiolabel incorporation remains unaffected for 7.5Ϫ10 min of the in vitro translation reaction, indicating that radiolabel incorporation is regulated by translation elongation. The rate of [ 35 S]Met incorporation into D1-protein can be increased by addition of exogenous ATP to the in vitro translation reactions; however, ATP cannot replace light, and at physiological concentrations of stromal ATP (40 µM), the rate is at least 25-fold higher in the light than in darkness. This indicates that translation elongation is arrested in darkness. Separation of translation-elongation reactions into polysome-bound and membrane-integrated D1 proteins demonstrates that the rate of translation elongation is higher in the presence of light. In the light, less time is required to transiently radiolabel a D1 translation intermediate of about 17 kDa and to chase the translation intermediate into mature D1 protein.We propose that light regulates the enzymatic activity of the translation-elongation process in chloroplasts.Keywords : chloroplast; translation elongation ; D1.Posttranscriptional regulation of gene expression in higher plant plastids has been intensively studied [1,2]. Studies of gene expression during plastid development and in mature chloroplasts have provided evidence for a light-induced regulation of translation [3Ϫ6]. Specifically, the light-regulated translation of the psbA mRNA encoding the photosystem II reaction-center protein D1 has been investigated in detail.In etioplasts isolated from 4-day-old dark-grown barley seedlings, the psbA mRNA was shown to be continuously translated ; however, the D1 protein was degraded in the absence of chlorophyll (Chl) and stabilized by binding of de novo synthesized Chl [7Ϫ10]. When 7Ϫ8-day-old dark-grown barley was illuminated, accumulation of D1 was dependent on developmentally regulated cytoplasmic factors and the psbA mRNA was found to be regulated on the level of translation initiation [11Ϫ 14]. In the light, analysis of ribosome distribution on psbA mRNA supported an accumulation of ribosomes at the site of initiation, but also revealed that ribosomes pause during translation elongation [14Ϫ16]. Pausing of ribosomes was speculated to improve the efficiency of Chl binding to D1 nascent chains or to enable integration of the D1 protein into the etioplast membranes during greening [9,15].In plastids isolated from transgenic tobacco leaves and in Chlamydomonas reinhardtii, light-dependent accumulation of D1 has been ascribed to regulation at the 5′-untranslated region of the psbA mRNA [17Ϫ19] ; structured RNA elements were characterized to be responsible for translation initiation and proteins were identified that specifically bind to the 5′-untranslated region during translation initiation [17,20,21] nas, ADP-dependent ...

SummaryAlthough several induction systems have been described for plants containing transgenes in the nucleus, to date there is only one method for controlling transgene expression in plastids. This consists of chemical induction of a nuclear gene and import of the gene product into plastids, so that transformation of two cellular compartments is required. Here we describe a system for external control of plastid gene expression which is based entirely on plastid components and can therefore be established in a single transformation step. Our system uses modified promoters containing binding sites for the bacterial lac repressor. Chemical induction can be made with intact plants or after harvesting, which provides ecological and economic benefits.