Lack of conservation of editing sites in mRNAs that encode subunits of the NAD(P)H dehydrogenase complex in plastids and mitochondria of Arabidopsis thaliana.

RNA editing in the plastids and mitochondria of higher plants involves C to U conversion of specific nucleotides in the mRNA. This leads to the synthesis of proteins that are different from those predicted by the DNA sequence. Editing appears to have arisen at about the same time in both plastids and mitochondria, suggesting a common evolutionary origin. The problem we address here is whether or not there has been co-evolution of the editing systems in the two organelles. Our test system was editing of the Arabidopsis thaliana mRNAs for ndhB and nad2, and for ndhD and nad4, which encode homologous subunits of the plastid and mitochondrial NAD(P)H dehydrogenases, respectively. The editing sites in the Arabidopsis nad2 and nad4 mRNAs have previously been determined and we report here 19 editing sites in eight mRNAs in Arabidopsis plastids. Out of these, eight sites are localized in the ndhB mRNA. In its mitochondrial counterpart, nad2, 31 editing sites are present, none of which are shared with the ndhB gene. The Arabidopsis ndhD mRNA is edited at four positions, only one of which is shared by its mitochondrial homologue, nad4, which contains 32 editing sites. These findings suggest that, although editing in the two organelles may have derived from a single system, there is no significant conservation of editing sites in cognate mRNAs in plastids and mitochondria.

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system.

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

Expression of bar in the plastid genome confers herbicide resistance.

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs.

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine-Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5'-untranslated region (5'-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5'-UTR and DB region promotes accumulation of NPTII up to approximately 16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield ( approximately 23% of TSP) was obtained from a construct in which the T7g10 5'-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

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.

Engineering of the rpl23 gene cluster to replace the plastid RNA polymerase alpha subunit with the Escherichia coli homologue.

The Escherichia coli RNA polymerase (RNAP) alpha, beta, and beta' core subunits are evolutionarily conserved among bacteria and plastids, and the plastid specificity factors form a functional holoenzyme with the E. coli core. To investigate whether the E. coli core subunits may form a functional hybrid enzyme with the plastid core subunits, we replaced the tobacco plastid RNAP alpha subunit gene (rpoA) with the E. coli alpha subunit gene by targeted gene insertion. The transplastomic tobacco plants look similar to tobacco rpoA deletion mutants in that they are chlorophyll-deficient and nonphotoautotrophic. In addition, they lack transcripts from promoters recognized by the E. coli-like plastid RNA polymerase. These results indicate that evolutionary conservation between the E. coli and plastid RNA polymerase alpha subunits is insufficient to allow substitution of the tobacco alpha subunit with its bacterial counterpart. Interestingly, the cyanobacterial alpha subunits are as different as the E. coli alpha subunits; and therefore it is unlikely that replacement of the tobacco alpha subunit with cyanobacterial alpha subunits would yield a functional enzyme. Replacement of plastid rpoA with the E. coli RNA polymerase alpha subunit gene represents the first engineering of a plastid operon in higher plants.

Conservation of RNA editing between rice and maize plastids: are most editing events dispensable?

The extent of conservation of RNA editing sites in the plastid genome of rice was determined by comparing the genomic sequence with that of the cDNA. The presence of a T in the cDNA predicted to be a C by the DNA sequence of the plastid genome, indicated C to U editing. In the 11 plastid transcripts of rice a total of 21 editing sites were found. In maize, a closely related grass species, 26 editing sites have been reported in 13 plastid transcripts. Most editing sites are conserved between the two species, although differences in RNA editing were found at eight sites. In seven cases the T was already encoded at the DNA level, eliminating the requirement for RNA editing. In one case (rpoB, codon 206) the RNA sequence was conserved between the two species, but the mRNA is still not edited in rice. It appears that, although evolutionarily conserved, RNA editing is essential only for a few plastid editing sites. Information about RNA editing in rice plastids will facilitate the design of plastid vectors with broad applicability in grass species.

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.

Plastome engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase in tobacco to form a sunflower large subunit and tobacco small subunit hybrid.

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters.

We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1). Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral. Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core. The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.

The phage-type PclpP-53 plastid promoter comprises sequences downstream of the transcription initiation site.

The existence of a phage-type plastid transcription machinery (NEP), related to the mitochondrial RNA polymerase, has been recognized only recently. Here we report the cis sequences required for transcription initiation by the phage-type enzyme. The promoter chosen for the study, PclpP-53, is well expressed in tobacco leaves, unlike most NEP promoters. Promoter definition was carried out in vivo , in transplastomic tobacco plants expressing a uidA reporter gene from PclpP-53 promoter derivatives. We report here that sequences from -5 to +25 (relative to the transcription initiation site) are sufficient to support specific transcription initiation. Requirement of sequences downstream of the transcription initiation site contrasts with mitochondrial promoters, which have conserved sequences predominantly upstream. The promoter defined here is conserved in liverworts and conifers, indicating that the phage-type transcription machinery appeared in plastids early on during the evolution of land plants. The PclpP-53 promoter sequences are present in rice but do not function, suggesting that PclpP-53 recognition specificity is absent in some monocots.

rbcL Transcript levels in tobacco plastids are independent of light: reduced dark transcription rate is compensated by increased mRNA stability.

The plastid rbcL gene, encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase, in higher plants is transcribed from a sigma70 promoter by the eubacterial-type RNA polymerase. To identify regulatory elements outside of the rbcL -10/-35 promoter core, we constructed transplastomic tobacco plants with uidA reporter genes expressed from rbcL promoter derivatives. Promoter activity was characterized by measuring steady state levels of uidA mRNA on RNA gel blots and by measuring promoter strength in run-on transcription assays. We report here that the rbcL core promoter is sufficient to obtain wild-type rates of transcription. Furthermore, the rates of transcription were up to 10-fold higher in light-grown leaves than in dark-adapted plants. Although the rates of transcription were lower in the dark, rbcL mRNA accumulated to similar levels in light-grown and dark-adapted leaves. Accumulation of uidA mRNA from most rbcL promoter deletion derivatives directly reflected the relative rates of transcription: high in the light-grown and low in the dark-adapted leaves. However, uidA mRNA accumulated to high levels in a light-independent fashion as long as a segment encoding a stem-loop structure in the 5' untranslated region was included in the promoter construct. This finding indicates that lower rates of rbcL transcription in the dark are compensated by increased mRNA stability.

RNA polymerase subunits encoded by the plastid rpo genes are not shared with the nucleus-encoded plastid enzyme.

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the beta-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP alpha-, beta'-, and beta"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Transcription from heterologous rRNA operon promoters in chloroplasts reveals requirement for specific activating factors.

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a sigma70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.

Plastid promoter utilization in a rice embryogenic cell culture.

Plastid promoter utilization was characterized in rice by mapping transcript 5'-ends in samples derived from leaves and cultured embryogenic cells. We have found that rbcL, atpB and the rRNA operon are transcribed by the plastid-encoded plastid RNA polymerase (PEP), while clpP is transcribed by the nucleus-encoded plastid RNA polymerase (NEP) in both chloroplasts and the non-green plastids of embryogenic cultured cells. This finding is in contrast to reports on BY2 tobacco, in which NEP promoter activity in cultured cells was enhanced relative to leaves, facilitating identification of NEP promoters which are undetectable in chloroplasts. Therefore, it appears that activation of plastid NEP promoters in rice is not essential for adaptation to cell culture.

Mapping of promoters for the nucleus-encoded plastid RNA polymerase (NEP) in the iojap maize mutant.

Plastid genes of higher plants may be transcribed by the plastid-encoded or the nucleus-encoded plastid RNA polymerases (PEP or NEP). The objective of this study was to identify NEP promoters in maize. To separate the NEP and PEP transcription activity, NEP promoter mapping was carried out in the iojap maize mutant which lacks the PEP. We report here that atpB, an ATPase subunit gene has promoters for both NEP and PEP, while clpP, a protease subunit gene, and the rpoB operon, encoding three PEP subunit genes, are exclusively transcribed from NEP promoters. The maize NEP promoters share sequence homology around the transcription initiation site, including the ATAGAATA/GAA loose consensus identified for tobacco, suggesting conservation of the NEP transcription machinery between monocots and dicots.

Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes.

The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.

A negative selection scheme based on the expression of cytosine deaminase in plastids.

The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil. CD is present in prokaryotes and in many eukaryotic micro-organisms, but is absent in higher plants. 5-fluorocytosine (5FC) is metabolized in CD-expressing cells, causing cellular death. A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD. This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.