Analysis of cis-acting expression determinants of the tobacco psbA 5’UTR in vivo

Chloroplast gene expression is predominantly regulated at the posttranscriptional levels of mRNA stability and translation efficiency. The expression of psbA, an important photosynthesis-related chloroplast gene, has been revealed to be regulated via its 5’- untranslated region (UTR). Some cis-acting elements within this 5’UTR and the correlated trans-acting factors have been defined in Chlamydomonas. However, no in vivo evidence with respect to the cis-acting elements of the psbA 5’UTR has been so far achieved in higher plants such as tobacco. To attempt this, we generated a series of mutants of the tobacco psbA 5’UTR by base alterations and sequence deletions, with special regard to the stem-loop structure and the putative target sites for ribosome association and binding of nuclear regulatory factors. In addition, a versatile plastid transformation vector pKCZ with an insertion site in the inverted repeat region of the plastid genome was constructed. In all constructs, the psbA 5’UTR (Wt or modified) was used as the 5’ leader of the reporter gene uidA under control of the same promoter, Prrn, the promoter of the rRNA operon. Through biolistic DNA delivery to tobacco chloroplasts, transplastomic plants were obtained. DNA and RNA analyses of these transplastomic plants demonstrated that the transgenes aadA and uidA had been correctly integrated into the plastome at the insertion site, and transcribed in discrete sizes. Quantitative assays were also done to determine the proportion of intact transplastome, the uidA mRNA level, Gus activity, and uidA translation efficiency. The main results are the following: 1) The insertion site at the unique MunI between two tRNA genes (trnR-ACG and trnNGUU) is functional. Vector pKCZ has a large flexibility for further DNA manipulations and hence is useful for future applications. 2) The stem-loop of the psbA 5’UTR is required for mRNA stabilisation and translation. All mutants related to this region showed a 2~3 fold decrease in mRNA stability and a 1.5~6 fold reduction in translation efficiency. The function of this stem-loop depends on its correct sequence and secondary conformation. 3) the AU-box of the psbA 5’UTR is a crucial translation determinant. Mutations of this element almost abolished translation efficacy (up to 175-fold decrease), but did not significantly affect mRNA accumulation. The regulatory role of the AU-Box is sequencedependent and might be affected by its inner secondary structure. 4) The internal AUG codon of the psbA 5’UTR is unable to initiate translation. An introduction of mRNA translatability from this codon failed to direct the translation of reporter uidA gene, overriding the mutation of the AU-Box. 5) The 5’end poly(A) sequence does not confer a distinct regulatory signal. The deletion of this element only insignificantly affected mRNA abundance and translation. However, this mutation might slightly disturb the conformation of the stem-loop, resulting in a moderate decrease in translation efficiency (~1.5 fold). 6) The SD(Shine-Dalgarno)-like RBS (ribosome binding site) of the psbA 5’UTR appears to be an indispensable element for translation initiation. Mutation of this element led to a dramatically low expression of the uidA gene as seen by Gus staining. 7) The 5’end structural sequence of the rbcL 5’UTR does not convey a high mRNA stabilising effect to the psbA 5’UTR in a cycling condition of the light and the dark. Their distinct roles appear to be involved in darkness adaptation. Furthermore, with respect to the overall regulatory function of the psbA 5’UTR, two models are proposed, i.e. dual RBS-mediated translation initiation, and cpRBPs-mediated mRNA stability and translation. The mechanisms for mRNA stabilisation entailed by the rbcL 5’UTR are also discussed. Direct repeat-mediated transgene loss after chloroplast transformation and other aspects related to the choice of insertion site and plastid promoter are also analysed.