In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency

Abstract

In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation ef®ciency Texas A&M University, College Station, Texas, USA Summary 5¢ and 3¢ untranslated regions (UTRs) of plastid RNAs act as regulatory elements for post-transcriptional control of gene expression. Polyethylene glycol-mediated plastid transformation with UTR±GUS reporter gene fusions was used to study the function of the psbA, rbcL and rpl32 UTRs in vivo. All gene fusions were expressed from the same promoter, i.e. the promoter of the 16S-rRNA gene, such that variations in RNA and protein levels would be due to the involved UTR elements alone. Transgenic tobacco lines containing different combinations of UTRs showed ®vefold variation in the uidA±mRNA level (RNA stability) and approximately 100-fold differences in GUS activity, a measure of translation activity. The rbcL 5¢-UTR conferred greater mRNA stability than the psbA 5¢-UTR on uidA transcripts. In contrast, the psbA 5¢-UTR enhanced translation of GUS to a much greater extent compared to the rbcL 5¢-UTR. The psbA 5¢-UTR also mediated light-induced activation of translation which was not observed with other constructs. Deletion mutagenesis of an unanalysed terminal sequence element of the psbA 5¢-UTR resulted in a twofold drop in uidA-mRNA level and a fourfold decrease in translation ef®ciency. Exchange of 3¢-UTRs results in up to ®vefold changes of mRNA levels and does not signi®cantly in¯uence translation ef®ciency. The mechanical impacts of these results on plastid translation regulation are discussed.

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

“…It has previously been shown that genes under the control of the psbA promoter/5#UTR achieve very high levels of expression (Fernandez San-Millan et al, 2003;Dhingra et al, 2004;Watson et al, 2004). The 5#UTR has been hypothesized to enhance translation of proteins under its control (Eibl et al, 1999). The aadA gene confers spectinomycin resistance for selection of transformed shoots (GoldschmidtClermont, 1991).…”

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

“…Because reporter protein levels do not always correlate with steady-state transcript abundance, it is evident that translation is a crucial control step for optimizing protein production. Several studies have revealed a minimal effect of 3′UTR sequences on chloroplast gene translation [42] although, recently, the petD 3′UTR was shown to influence reporter protein expression [43]. By contrast, many 5′UTRs mediate enhanced translation of downstream open-reading-frames [44].…”

“…By contrast, many 5′UTRs mediate enhanced translation of downstream open-reading-frames [44]. One of the most effective of these, the tobacco psbA 5′UTR, directs strong, light-regulated, translation of chimeric transcripts regardless of which coding sequence, 3′UTR or promoter it is combined with [42].…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…The junction point of the large single-copy region (LSC) and IR-A of the tobacco plastome (Shinozaki et al, 1986) are shown. Gene abbreviations can be found in Table I Staub and Maliga, 1994a;Eibl et al, 1999). However, researchers still routinely used regulatory elements including promoters and untranslated regions with homology to sequences within the host plastome (Verma and Daniell, 2007), often because these were the only sequences readily available for use.…”

“…While these expression levels are too high for most metabolic engineering strategies for the production of chemicals, fuels, or materials without creating unwanted stress on the host plant, expression levels can be somewhat controlled by the choice of regulatory elements flanking the transgenes. Plastid gene expression is regulated to a large extent at the posttranscriptional level (Sugita and Sugiura, 1996;Stern et al, 1997;Bruick and Mayfield, 1999;Dubald et al, 2008), and 5# and 3# untranslated regions (UTRs) have been shown to impact translational efficiency and mRNA stability, respectively (Eibl et al, 1999). However, these UTR sequences, as well as any other sequences with significant homology to the host's plastome, must be used with care, since they can cause unwanted rearrangements (Svab and Maliga, 1993;Staub and Maliga, 1994a;Rogalski et al, 2006Rogalski et al, , 2008McCabe et al, 2008;Zhou et al, 2008;Gray et al, 2009).…”