Plastid RNA Editing Research Articles

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids.

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.

Introducing an RNA editing requirement into a plastid-localised transgene reduces but does not eliminate functional gene transfer to the nucleus

In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum (tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines (tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene (neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment.

Unparalleled GC content in the plastid DNA of Selaginella

One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date-the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.

AtECB2, a pentatricopeptide repeat protein, is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana

Chloroplast biogenesis is a complex process in higher plants. Screening chloroplast biogenesis mutants, and elucidating their molecular mechanisms, will provide insight into the process of chloroplast biogenesis. In this paper, we obtained an early chloroplast biogenesis mutant atecb2 that displayed albino cotyledons and was seedling lethal. Microscopy observations revealed that the chloroplast of atecb2 mutants lacked an organized thylakoid membrane. The AtECB2 gene, which is highly expressed in cotyledons and seedlings, encodes a pentatricopeptide repeat protein (PPR) with a C-terminal DYW domain. The AtECB2 protein is localized in the chloroplast, and contains a conserved HxEx(n)CxxC motif that is similar to the activated site of cytidine deaminase. The AtECB2 mutation affects the expression pattern of plastid-encoded genes. Immunoblot analyses showed that the levels of photosynthetic proteins decreased substantially in atecb2 mutants. Inspection of all reported plastid RNA editing sites revealed that one editing site, accD, is not edited in atecb2 mutants. Therefore, the AtECB2 protein must regulate the RNA editing of this site, and the dysfunctional AccD protein from the unedited RNA molecules could lead to the mutated phenotype. All of these results indicate that AtECB2 is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana.

The Arabidopsis gene YS1 encoding a DYW protein is required for editing of rpoB transcripts and the rapid development of chloroplasts during early growth

Virescence, a phenotype in which leaves green more slowly than usual, is recognized to play a role in protection from photo-oxidative damage before healthy chloroplasts are developed. The elucidation of the molecular mechanisms underlying virescence will provide insights into how the development of chloroplasts is controlled. In this study, we find that knockout alleles of Yellow Seedlings 1 (YS1) in Arabidopsis lead to a virescent phenotype, which disappears by 3 weeks after germination. The ys1 mutation resulted in marked decreases in photosynthetic capacity and photosynthetic pigment complexes, and disturbed ultrastructure of thylakoid membranes in 8-day-old seedlings. However, cotyledons of ys1 seedlings pre-treated in the dark for 5 days turn green almost as fast as the wild type in light, revealing that the developmental defects in ys1 are limited to the first few days after germination. Inspection of all known plastid RNA editing and splicing events revealed that YS1 is absolutely required for editing of site 25992 in rpoB transcripts encoding the beta subunit of the plastid-encoded RNA polymerase (PEP). YS1 is a nuclear-encoded chloroplast-localized pentatricopeptide repeat protein differing from previously described editing factors in that it has a C-terminal DYW motif. A defect in PEP activity is consistent with the changes in plastid transcript patterns observed in ys1 seedlings. We conclude that the activity of PEP containing RpoB translated from unedited transcripts is insufficient to support rapid chloroplast differentiation.

Amino acid sequence variations in Nicotiana CRR4 orthologs determine the species-specific efficiency of RNA editing in plastids

In flowering plants, RNA editing is a posttranscriptional process that converts specific C to U in organelle mRNAs. Nicotiana tabacum is an allotetraploid species derived from the progenitors of Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana species have been used as a model for understanding the mechanism and evolution of RNA editing in plastids. In Nicotiana species, the ndhD-1 site is edited to create the translational initiation codon of ndhD that encodes a subunit of the NAD(P)H dehydrogenease (NDH) complex. An analysis of this RNA editing revealed that editing efficiency in N. tomentosiformis is lower (15%) than that in N. tabacum (42%) and N. sylvestris (37%). However, this level of editing is sufficient for accumulating the NDH complex and its activity. The heterogous complementation of Arabidopsis crr4-3 mutant, in which RNA editing of ndhD-1 is completely impaired, with CRR4 orthologous genes derived from Nicotiana species suggested that the reduction in editing efficiency in N. tomentosiformis is caused by amino acid variations accumulating in CRR4.

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein

Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts.

Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.

A Pentatricopeptide Repeat Protein Is a Site Recognition Factor in Chloroplast RNA Editing

In higher plants, RNA editing is a post-transcriptional process that converts C to U in organelle mRNAs. We have previously shown that an Arabidopsis thaliana crr4 mutant is defective with respect to RNA editing for creating the translational initial codon of the plastid ndhD gene (the ndhD-1 site). CRR4 contains 11 pentatricopeptide repeat motifs but does not contain any domains that are likely to be involved in the editing activity. The green fluorescent protein fused to the putative transit peptide of CRR4 targeted the plastid. The recombinant CRR4 expressed in Escherichia coli specifically bound to the 25 nucleotides of the upstream and the 10 nucleotides of the downstream sequences surrounding the editing site of ndhD-1. The target C nucleotide of this editing is not essential for the binding of CRR4. Taken together with the genetic evidence, we conclude that the pentatricopeptide repeat protein CRR4 is a sequence-specific RNA-binding protein that acts as a site recognition factor in plastid RNA editing.

Maintenance of plastid RNA editing activities independently of their target sites

RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell.

Pre-screening plastid transgene expression cassettes in Escherichia coli may be unreliable as a predictor of expression levels in chloroplast-transformed plants

A di-cistronic expression cassette ( Hb) encoding the α- and β-subunits of human adult hemoglobin, under the transcriptional control of a phage T7 promoter, was introduced into the tobacco plastid genome. The resulting chloroplast-transformed line, Hb1, was crossed with a nuclear-transformed line, PR-T7A, expressing a salicylic acid-inducible plastid-targeted T7 RNA polymerase in order to activate Hb transcription. Even in the absence of induction, Hb transcripts were expressed constitutively in Hb1xPR-T7A progeny plants. Treatment of leaves with salicylic acid resulted in an additional five-fold increase in Hb transcript levels. However, despite the very high-level of Hb transcript accumulation in Hb1xPR-T7A plants and the fact that the Hb expression cassette directed the synthesis of hemoglobin in Escherichia coli, recombinant hemoglobin did not accumulate at levels detectable by immunoblot analysis in chloroplast-transformed plants. Furthermore, Hb transcripts present in total leaf RNA isolated from Hb1xPR-T7A plants directed hemoglobin synthesis in an E. coli-derived in vitro translation system thus excluding the possibility that Hb mRNA might have been rendered untranslatable by the plastid RNA editing machinery.

RNA editing in ribosome-less plastids of iojap maize.

In maize chloroplasts, 28 C-to-U editing events have been identified in the transcripts of 14 different genes. The iojap mutant of maize, which lacks chloroplast ribosomes, affords the opportunity to examine whether any chloroplast translation products are required for the editing of any of the 28 sites. Furthermore, the mode of action of the IOJAP protein itself is unknown, so we explored the possibility that homozygous ij1/ ij1 plants are defective in RNA editing. Current knowledge of RNA editing in chloroplasts indicates the existence of site-specific factors responsible for recognizing C targets of editing, but the factors have not yet been identified and their encoding genes are unknown. Our results indicate that all 28 editing sites can be recognized and processed in ribosome-less plastids. Transcripts of rpoB are more abundant and more highly edited in iojap mutants. The editing site in rpl2, which creates the mRNA start codon, is the most severely affected in homozygous ij/ ij plants, but nevertheless exhibits at least 10% editing in all mutant lines. Reduced editing of rpl2 may be an indirect effect of reduced splicing, rather than a defect caused by the iojap mutation. We conclude that neither the IOJAP protein nor chloroplast translation products are required for editing any of the 28 C targets of editing in maize chloroplast RNAs.

Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon.

RNA editing in higher plant plastids is a post- transcriptional RNA maturation process changing single cytidine nucleotides into uridine. In the ndhD transcript of tobacco and several other plant species, editing of an ACG codon to a standard AUG initiator codon is believed to be a prerequisite for translation. In order to test this assumption experimentally, we have analyzed the editing status of ndhD mRNA species in the process of translation. We show that unedited ndhD transcripts are also associated with polysomes in vivo, suggesting that they are translated. This surprising finding challenges the view that ACG to AUG editing is strictly required to make the ndhD message translatable and raises the possibility that ACG can be utilized as an initiator codon in chloroplasts. In addition, we have mapped the termini of the ndhD transcript and discovered a novel form of RNA processing. Unexpectedly, we find that highly specific sequences are added to the 3' end of the ndhD mRNA at high frequency. We propose a model in which these sequences are added by the successive action of a CCA-adding enzyme (tRNA nucleotidyltransferase) and an RNA-dependent RNA polymerase (RdRp) activity. The presence of an RdRp activity may have general implications also for other steps in plastid gene expression.

Is clustering of plastid RNA editing sites a consequence of transitory loss of gene function? – Implications for past environmental and evolutionary events in plants

Abstract Editing changes individual nucleotides in RNA thereby distinguishing the mature transcript sequence from the corresponding genomic sequence. Most editing events in chloroplasts change cytosine to uracil bases at specific positions in transcripts. In hornwort and fern species uracil to cytosine conversions also take place. RNA editing in plastids probably emerged among the first land plants and affects different genes of different plants in different positions. A significant clustering of editing sites in certain genes is found within related taxa. The clustering is particularly evident in ndh genes in angiosperms, where editing sites account for about 50% of total (near 30) editing sites of plastids and are rather similar in a large number of species. In gymnosperms, ndh genes are edited to a much lower extent, and the few editing sites identified in bryophytes are different from those of angiosperms. On the basis of these data and the role of ndh genes in the protection against photooxidative stress, we make the following hypothesis for the origin and evolution of editing sites: (i) primary mutations accumulated in genes which became dispensable for plants in certain ecosystems; (ii) under changing environments, the functionality of these genes was recovered in some of the descendants by transcript editing; and (iii) secondary mutations led to a subsequent and gradual disappearance of editing sites. Evidence and further tests for this hypothesis are discussed.

RNA editing in higher plant plastids: oligoribonucleotide SSCP analysis allows the proof of base conversion directly at the RNA level.

Plastid RNA editing of a number of transcripts at specific sites changes genomically encoded cytidines to nucleosides, which act like uridines in RT-PCR analyses. To study plastid-editing directly at the RNA level, we established a single-strand conformational polymorphism assay for the discrimination of small RNA molecules. The electrophoretic mobility of a oligoribonucleotide resulting from a RNase T1-digested and edited plastid mRNA was shown to be identical with a control RNA molecule containing a uridine at the editing site, whereas the unedited RNA behaved like a RNA molecule containing a cytidine at the respective position.

Participation of nuclear genes in chloroplast gene expression

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

RNA editing in an untranslated region of the Ginkgo chloroplast genome.

mRNAs in plant cell organelles can be subject to RNA editing, an RNA processing step altering the identity of single nucleotide residues. In higher plant chloroplasts, editing proceeds by C-to-U conversions at highly specific sites. All known plastid RNA editing sites are located in protein-coding regions and, typically, change the coding properties of the mRNA. To gain more insight into the evolution of editing, we have determined the molecular structure and RNA editing pattern of the psbE operon of the primitive seed plant Ginkgo biloba. We report here the identification of altogether four sites of C-to-U editing, two of which are unique to Ginkgo and have not been found in other species. Surprisingly, one of the sites is located in an intercistronic spacer, thus being the first chloroplast editing site detected outside a protein-coding region. This indicates that the plastid editing machinery can operate also in untranslated regions and without having apparent functional consequences.

Transfer of plastid RNA-editing activity to novel sites suggests a critical role for spacing in editing-site recognition.

RNA editing in higher plant plastids alters mRNA sequences by C-to-U conversions at highly specific sites through an unknown mechanism. To elucidate how the cytidine residues to be edited are specifically recognized and distinguished from other cytidines in close proximity, we have changed in vivo the distances of two plastid RNA-editing sites from their essential upstream cis-acting sequence element. Analysis of RNA editing in transgenic chloroplasts revealed that reduction of this distance by 1 nt entirely abolishes RNA editing. Surprisingly, deletions or combinations of deletional and point mutations that shift a heterologous cytidine residue in the same distance from the upstream cis-element as the editing site in the wild type result in transfer of the RNA-editing activity to the heterologous cytidine whereas the wild-type site remains unedited. Our results suggest that the molecular identity of at least some editing sites in the chloroplast genome is defined by their distance from an essential upstream sequence element.

Analysis of RNA Editing in Plastids

Chloroplast mRNAs can be subject to posttranscriptional pyrimidine-to-pyrimidine conversions at highly specific sites. This RNA modification mechanism shows a high degree of similarity to plant mitochondrial editing but differs markedly from, and is most likely evolutionarily unrelated to, all other RNA editing systems. The study of RNA editing processes in chloroplasts has been largely hampered by the lack ofin vitroediting systems; however, considerable insights into the recognition mechanisms of individual editing sites have come fromin vivoapproaches. Chloroplast transformation proved to be a particularly useful tool to study plastid RNA editing. In this article, specific methods for the analysis of chloroplast RNA editing are discussed. Detailed experimental procedures are provided for (i) the purification of chloroplasts and (ii) the stable genetic transformation of higher plant plastids.

Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: a role for plastid translation in RNA editing.

RNA editing in higher plant plastids changes single cytidine residues to uridine through an unknown mechanism. In order to investigate the relation of editing to physiological processes and to other steps in plastid gene expression, we have tested the sensitivity of chloroplast RNA editing to heat shock and antibiotics. We show that heat shock conditions as well as treatment of plants with prokaryotic translational inhibitors can inhibit plastid RNA editing. Surprisingly, this inhibitory effect is confined to a limited number of plastid editing sites suggesting that some site-specific factor(s) but none of the general components of the plastid RNA editing machinery are compromised. Contrary to previous expectations, our results provide evidence for a role of plastid translation in RNA editing.