Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts

The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.     

Removal of antibiotic resistance genes from transgenic tobacco plastids

Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.     

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.     

Construction of a promoter-probe shuttle vector for Escherichia coli and brevibacteria

We constructed a promoter-probe vector, pJUP05, for brevibacteria and Escherichia coli based on the promoterless neomycin-resistance (neoR) gene from Tn5. This gene confers resistance to the aminoglycosides, kanamycin and neomycin. The promoter of the neoR gene was deleted and replaced by a suitable multiple cloning site. There are translation stop codons in all three reading frames upstream from the neoR gene. The plasmid contains functional origins of DNA replication for both brevibacteria and E. coli, and permits selection for chloramphenicol- and/or ampicillin-resistance markers.     

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.