A demanding task in the generation of transgenic plants is the option of simultaneously introducing two or more genes into an organism. Conventional approaches require the combination of separate expression cassettes each containing a promoter and terminator region framing the gene of interest. In contrast, plastids are thought to offer an unique option of combining multiple ORFs under the control of one promoter, yielding a polycistronic transcript from which translation can be initiated independently (Staub and Maliga 1995). However, except for a few examples like the cry2Aa2 operon from Bacillus thuringiensis (De Cosa et al. 2001; Quesada-Vargas et al. 2005), this technique has not been utilized for the simultaneous production of two or more recombinant proteins so far. This is probably due to the hitherto unpredictable secondary structure interactions in polycistronic transcripts, which determine the translatability and processing into monocistronic mRNAs and subsequently result in poor protein accumulation. Although the similarities between transcription and translation in bacteria and in plastids are striking, one cannot generally extrapolate results obtained in bacteria to plastids. For instance, an operon encoding for hemoglobin a- and p-subunits, which worked well in E. coli, did not lead to detectable expression when integrated into plastids (Magee et al. 2004). A recent study identified a so-called intercistronic expression element (IEE), a short sequence that mediates the cleavage of a polycistronic precursor into stable monocistronic transcripts (Zhou et al. 2007). It will be interesting to see whether this novel element leads to concerted high-level expression of recombinant proteins.
Another challenge is the regulation of gene expression in plastids, as it is state of the art in E. coli. It would be highly desirable to avoid deleterious or toxic effects of recombinant proteins on plant metabolism by initiating expression at will. Most promoters used so far are more or less constitutive (like the Prrn) or regulated by factors which could be hardly used for targeted expression initiation. In photosyn-thetically active chloroplasts especially the 5'-UTRs of several transcripts contribute to the regulation of translation. It has been shown that light regulates the translation of the psbA mRNA (Kim and Mullet 1994) while RNA levels are kept relatively constant (Shiina et al. 1998). However, since light cannot be withheld until the desired transgene expression needs to be initiated, it would be highly advantageous to have instead an inducible system at hand which relays on chemical or other physiological triggers. A sophisticated approach is to put the transgene under the control of the phage T7 promoter, which is per se not active in plastids. Expression can be only initiated by the appropriate T7 RNA polymerase, which needs to be introduced by genetic crossing with a plant line carrying the gene in the nucleus and fused to a plastid signal peptide (McBride et al. 1994). To add a regulatory element to this system, other studies used inducible nuclear promoters to control expression of the nuclear gene, e.g. the salicylic acid-inducible PR-1a promoter from tobacco (Magee et al. 2004) or an ethanol inducible promoter (Lossl et al. 2005). The disadvantage of this particular approach is that two subsequent transformations of different cellular compartments or genetic crossing of different transformants are necessary to obtain the final plant. Also the E.coli lac control system has been adopted for plastid expression. Therefore, the lacI repressor needed to be co-expressed together with the heterologous gene (gfp) under control of a modified rrn/ T7g10 promoter inside the plastids (Muhlbauer and Koop 2005). Spraying of plants with isopropyl thiogalactoside (IPTG) indeed induced GFP-formation, but it needs to be established whether this method is applicable on a large scale.
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