MBE Advance Access originally published online on April 9, 2008
Molecular Biology and Evolution 2008 25(7):1405-1414; doi:10.1093/molbev/msn084
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Research Articles |
Organellar RNA Editing and Plant-Specific Extensions of Pentatricopeptide Repeat Proteins in Jungermanniid but not in Marchantiid Liverworts
Institut für Zelluläre und Molekulare Botanik, Abteilung Molekulare Evolution, Universität Bonn, Bonn, Germany
E-mail: volker.knoop{at}uni-bonn.de
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Abstract |
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The pyrimidine exchange type of RNA editing in land plant (embryophyte) organelles has largely remained an enigma with respect to its biochemical mechanisms, the underlying specificities, and its raison d’être. Apparently arising with the earliest embryophytes, RNA editing is conspicuously absent in one clade of liverworts, the complex thalloid Marchantiidae. Several lines of evidence suggest that the large gene family of organelle-targeted RNA–binding pentatricopeptide repeat (PPR) proteins plays a fundamental role in the sequence-specific editing of organelle transcripts. We here describe the identification of PPR protein genes with plant-specific carboxyterminal (C-terminal) sequence signatures (E, E+, and DYW domains) in ferns, lycopodiophytes, mosses, hornworts, and jungermanniid liverworts, one subclass of the basal most clade of embryophytes, on DNA and cDNA level. In contrast, we were unable to identify these genes in a wide sampling of marchantiid liverworts (including the phylogenetic basal genus Blasia)—taxa for which no RNA editing is observed in the organelle transcripts. On the other hand, we found significant diversity of this type of PPR proteins also in Haplomitrium, a genus with an extremely high rate of RNA editing and a phylogenetic placement basal to all other liverworts. Although the presence of modularly extended PPR proteins correlates well with organelle RNA editing, the now apparent complete loss of an entire gene family from one clade of embryophytes, the marchantiid liverworts, remains puzzling.
Key Words: RNA editing • PPR proteins • liverworts
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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RNA editing in plants was originally reported with 3 simultaneous papers documenting the exchange of cytidines into uridines in mitochondrial transcripts (Covello and Gray 1989
; Gualberto et al. 1989; Hiesel et al. 1989), shortly followed by a report on the same phenomenon in chloroplasts (Hoch et al. 1991). Subsequently, RNA editing was found to be operative in both organelles in all land plant (embryophyte) clades but suspiciously absent in the subclass of marchantiid liverworts (Malek et al. 1996; Freyer et al. 1997; Steinhauser et al. 1999). The reverse editing process, the reversion of uridines in the primary transcripts into cytidines very rarely observed in angiosperms (Schuster et al. 1990), is particularly pronounced in hornwort and fern organelle transcripts (Yoshinaga et al. 1996; Vangerow et al. 1999).
A series of papers have demonstrated that sequences of about 30 nt upstream of the site to be edited in the organelle transcripts are more relevant than downstream sequences to efficiently determine editing sites in both mitochondria (Kubo and Kadowaki 1997; Williams et al. 1998; Farre et al. 2001; Takenaka et al. 2004) and plastids (Bock et al. 1996; Chaudhuri and Maliga 1996; Chateigner-Boutin and Hanson 2002). Rates of RNA editing differ between transcripts in the 2 endosymbiotic organelles. In chloroplasts, on average 20–30 editing sites are found (Maier et al. 1995; Wakasugi et al. 1996; Hirose et al. 1999; Schmitz-Linneweber et al. 2002; Tillich et al. 2006), whereas the plant mitochondrial transcripts feature around 500 editing sites (Giege and Brennicke 1999; Knoop and Brennicke 1999; Notsu et al. 2002; Handa 2003; Mower and Palmer 2006). In certain species such as the quillwort Isoetes (lycopodiophytes), the number of RNA editing sites in a single given mRNA may easily exceed 70 sites (Malek et al. 1996; Grewe, Viehöver, Weisshaar, and Knoop unpublished data). Most RNA editing events are essential to express functional proteins by modifying the RNA sequences to reestablish conserved codon identities (Walbot 1991; Bock et al. 1994; Gray 1996; Hanson et al. 1996; Knoop 2004) or even by regenerating or removing stop or start codons (Hoch et al. 1991; Wintz and Hanson 1991; Maier et al. 1996; Kotera et al. 2005), respectively. Thus, RNA editing largely seems to be a posttranscriptional correction mechanism with vital importance for plants; yet, the precise process is still enigmatic.
After the establishment of in vitro or in organello editing assays to identify relevant protein factors biochemically (Araya et al. 1992; Hirose and Sugiura 2001; Miyamoto et al. 2002; Takenaka and Brennicke 2003, 2007; Choury et al. 2004; Neuwirt et al. 2005; Staudinger et al. 2005; Sasaki et al. 2006), 2 trans-factors were recently identified through molecular genetic approaches, which mediate recognition of specific editing sites in plastids (Kotera et al. 2005; Okuda et al. 2006, 2007). These proteins are members of a huge gene family in plants, the pentatricopeptide repeat (PPR) protein family first detected in Arabidopsis thaliana with about 450 members (Lurin et al. 2004; Saha et al. 2007). PPR proteins are defined by tandem arrays of 2–26 degenerate 
35 amino acid repeats (Small and Peeters 2000). Most of them are RNA-binding proteins targeted into the 2 semiautonomous endosymbiotic organelles (Lurin et al. 2004). In higher embryophytes like A. thaliana and Oryza sativa which contain high numbers of RNA editing sites in their organellar (mainly mitochondrial) transcripts, they form one of the largest and most diverse gene families (Lurin et al. 2004). Hence, they are perfect candidates to play a decisive role in RNA editing. PPR proteins are divided into 2 subfamilies, the P family with members composed of PPR motifs only and the PLS subfamily, also referred to as the plant combinatorial and modular protein family (Aubourg et al. 2000). Whereas few members of the P subfamily are identified in diverse eukaryotes, the PLS subfamily is restricted to embryophytes (Andres et al. 2007; Salone et al. 2007). Members of the PLS subfamily feature additional conserved carboxyterminal (C-terminal) domains, the E domain and the E+ and DYW domains as optional, successive extensions in this order (Lurin et al. 2004, fig. 1). The DYW domain is named after the nearly invariant C-terminal DYW tripeptide motif (aspartate–tyrosine–tryptophan). This conserved domain features several cysteine and histidine residues, which could be related to catalytic function (Aubourg et al. 2000) and, most importantly, has some distant similarity to cytidine deaminases (Salone et al. 2007), the type of enzyme most likely responsible for the conversion of cytidines into uridines in plant organelle RNAs (Blanc et al. 1995; Yu and Schuster 1995).
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First results seemed to indicate that the presence of DYW domains correlates well with the known phylogenetic distribution of RNA editing in embryophytes that is suspiciously absent among the complex thalloid marchantiid liverworts (Salone et al. 2007
). With the present study, we wished to 1) extend our previous work in its phylogenetic scope and corroborate, if possible, the phylogenetic data on PPR genes on transcript level, 2) explore whether DYW domains are similarly linked to other conserved domains in PLS-type proteins in basal most embryophyte clades known to show RNA editing activity, and 3) check whether DYW-type gene diversity correlates with RNA editing frequency as should be postulated for a model assuming a functional correlation. |
Materials and Methods |
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Total nucleic acids were prepared from plant material by the cetyl trimethyl ammonium bromide method. DNA and RNA were differentially fractionated in the presence of 3 M lithium acetate. The RNA fraction was treated with DNase I (Fermentas Life Sciences, St Leon-Rot, Germany) to remove potential vestiges of DNA. First-strand cDNA was synthesized using the RevertAid M-MulV Reverse Transcriptase kit (Fermentas Life Sciences). Primers DCHfor (5'-cacctcgagtgyiiigaytgycay-3') and CSCRDYW-Stop-rev (5'-tyaccartartcnctacaagaaca-3') bordering the DCH-DYW region were used for cDNA assays. To check cDNA quality, cDNA assays with primers H3upv2 (5'-atggcycgyacbaagcagac-3') and H3dov2 (5'-atgtcytttggcatgatggtgac-3') were performed. Upstream regions of the DCH-DYW domain were amplified by inverted polymerase chain reaction (iPCR) (Triglia et al. 1988
). Genomic DNA was cleaved alternatively with XmiI or Bsp143I (Fermentas Life Sciences). For circularization, 2 µg of the restriction fragments were self-ligated with 3 U T4 DNA ligase (Fermentas Life Sciences) in a 300 µl reaction volume. Ten microliters were used as template in the subsequent iPCR assay with primer combination DYWforward2 (5'-cattgttcttgcagagattattggt-3') and DCHrev2 (5'-gtatttaatggcagtgtggcagtc-3'). Fragments containing upstream regions of the DCH motif were reamplificated by a seminested PCR with primer combination DYWforward (5'-ggiiiitgykcitgysgigaytaytggt-3')/DCHreverse (5'-tgrcartciiircaiacicgiarrttytt-3') or CSCGDYWv2ifor (5'-ggnacntgctcttgtggrgaytactgg-3')/DCHv2irev (5'-gacaatcaccacagacnckaaggttctt-3'). Primer Ebox1for (5'-gshtaygtdytbhtrtcmaacatwta-3') combined with CSCR-DYW-Stop-rev was used to amplify further E/E+/DYW combinations of PPR genes from DNA and cDNA templates. PCR amplification assays contained 1 µl template DNA (10 ng–1 µg), 1x recommended PCR buffer, 1.5–2 mM MgCl2, 2 mM deoxynucleoside triphosphates, 0.2–0.4 µM of each primer, 1–1.25 U DNA polymerase, and double-distilled water up to 25 µl. Different commercially available thermostable DNA polymerase kits were used; Taq DNA Pol E (Genaxxon, Biberach, Germany), GoTaq DNA Pol (Promega, Mannheim, Germany), or the Triplemaster PCR System (Eppendorf, Hamburg, Germany). The majority of amplification assays included 3 min initial denaturation at 94 °C followed by 30 cycles each with 30 s denaturation at 94 °C, 30 s annealing at 50–35 °C (decreasing 0.5 K/cycle), and 3 min synthesis at 72 °C, additionally 10 cycles with annealing at 35 °C and a final step of synthesis for 7 min at 72 °C. In amplification assays using primer Eboxfor1, the circadian part was altered into 10 cycles with annealing at 45–35 °C (decreasing 1 °C/cycle) followed by 30 cycles with annealing temperature of 35 °C. PCR products were cloned into the pGEM-T Easy vector (Promega) and were commercially sequenced (Macrogen Seoul, Korea). Deduced protein sequences were aligned with MEGA 3.1 (Kumar et al. 2004) using the ClustalW algorithm and manually adjusted. |
Results |
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Starting from the End: The DYW Domain
Our studies were initiated from the particular plant-specific PLS subclade of PPR proteins carrying as an ultimate C-terminal extension the so called DYW domain, which is characterized by this tripeptide motif nearly invariably occurring as the ultimate amino acids in this subgroup of the PPR gene family in plants (fig. 1). We could amplify the C-terminal 39 amino acid segment of the DYW domain (the DCH-DYW region) by PCR using DNA templates from all major embryophyte clades: seed plants, ferns, fern allies, mosses, and hornworts, all of which are embryophyte groups that generally show organellar RNA editing. In case of the basal most clade of embryophytes, the liverworts, an interesting picture seemed to emerge: In contrast to the subclade of simple thalloid, jungermanniid liverworts and Haplomitrium mnioides, no products could be obtained for the complex thalloid marchantiid liverworts (Salone et al. 2007
), which also stand out by lacking RNA editing (Malek et al. 1996; Freyer et al. 1997; Steinhauser et al. 1999).
The DCH-DYW Region in Jungermanniid versus Marchantiid Taxa: cDNA Level
Although the amplification of the DCH-DYW region gave reasonable indications toward a borderline for its presence in jungermanniid and haplomitriid versus nonexistence in marchantiid liverworts, this may be due to experimental shortcomings or yet unrecognized genomic complexity of the latter, so we wished to confirm the observations. To prove that the DCH-DYW amplicons are parts of expressed genes and given that some plant DNA preparations can be notoriously difficult templates, we aimed to corroborate our results on the transcript level. Sufficient amount of RNA of adequate quality for cDNA synthesis was available for 7 jungermanniid, 6 marchantiid taxa, and H. mnioides (table 1). We accordingly used reverse transcriptase–polymerase chain reaction (RT-PCR) to amplify the DCH-DYW amplicon on the cDNA level. As a positive control for cDNA synthesis, the histone 3 (H3) gene was used as a low-copy nuclear gene reference. A RT-PCR assay is shown exemplarily in figure 2 with 4 marchantiid and 4 jungermanniid liverwort cDNA preparations used as templates, respectively. Whereas the histone H3 RT-PCR positive control product of expected size (372 bp) appeared in all cases, the expected DCH-DYW amplicons (139 bp) were only retrieved from the jungermanniid taxa (GenBank accession numbers: EU495621
[GenBank]
–22 and EU495627
[GenBank]
–49) and from H. mnioides (GenBank accession numbers: EU495623
[GenBank]
–26) but not from the marchantiid taxa (fig. 2). In general, PPR genes are only very weakly expressed, but notably the DCH-DYW amplicon products were retrieved proportional in amount to the H3 positive control among the jungermanniids, presumably just reflecting cDNA synthesis quality. However, DCH-DYW amplification products remained absent even in cases of very strong positive control H3 amplification signals from cDNA as in the cases of Conocephalum or Lunularia, respectively, among the Marchantiids (fig. 2).
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Extending the DCH-DYW Region: iPCR Approaches
The DYW domain is generally present as the ultimate extension of PPR proteins preceded by the E and the E+ domains in the DYW subgroup of PLS proteins known in A. thaliana and O. sativa (fig. 1). To clarify whether this protein structure would also be present in the jungermanniid liverworts, we used an iPCR strategy in which self-ligated (circularized) DNAs were used as templates for amplification with primers directed outward from the DCH-DYW region, followed by a second, seminested PCR to enhance specificity. Amplification products from the iPCR approach were successfully obtained for 5 jungermanniid liverworts: Diplophyllum albicans, Fossombronia cf. alaskana, Lepidogyna hodgsoniae, Scapania aequiloba, and Trichocolea tomentella. The lycopodiophyte Isoetes engelmannii was additionally included in our studies, given the demonstrated extraordinarily high frequency of RNA editing in this genus (Malek et al. 1996
) which would strongly postulate a PLS gene family in this taxon assuming a functional correlation. Although cloning and sequencing ultimately revealed that most of the iPCR products were not derived from bona fide circularization at the expected recognition sites of the enzymes used prior to self-ligation, a total of 18 loci from those 6 taxa were isolated, which clearly showed conserved sequences reaching into protein reading frames upstream of the DCH-DYW region (fig. 3). The clones expectedly cover coding regions upstream of the DYW domain to different extents but all clearly confirm the presence of conserved DYW, E+, and E motifs, respectively (fig. 3). The maximal extensions of reading frames were obtained with iPCR clones of the liverworts Diplophyllum and Fossombronia, which even reached beyond the complete E, E+, and DYW motifs into the further upstream PLS-coding regions. Most significantly, sequence comparisons clearly revealed that amino acid motifs previously found conserved among the angiosperms were likewise conserved among the liverwort sequences now retrieved, indicating conserved function in those amino acid residues (fig. 3).
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The E, E+, DYW Extension in Jungermanniid Liverworts
The iPCR approach clearly demonstrated that DYW-type PPR proteins are already present in the jungermanniid liverworts as the basal most clade in the plant lineage for which RNA editing had been identified. To further check for presence versus absence of this type of proteins in the 2 major clades of liverworts, we made use of the now available upstream sequences to design a new primer targeting the E domain (figs. 1 and 3). This primer was used in combination with the downstream DYW domain primer in PCR amplification. An exemplary PCR result is shown in figure 4 for DNA templates from 6 jungermanniid species, H. mnioides, and 7 marchantiid species, respectively. Amplification products of the expected size of 507 bp were obtained with this new approach for several gymnosperms, ferns, hornworts, mosses, jungermanniid, and haplomitriid template DNAs, whereas no products could be retrieved for any of the marchantiid species investigated (in total from 12 marchantiid DNA preparations, table 1). Notably, the positive PCR results suggest absence of intron sequences in the nonangiosperm clades as usually observed for the PPR protein genes in the flowering plants as well (Lurin et al. 2004
).
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We again wished to corroborate the DNA-based observations on the cDNA level and performed RT-PCR experiments with cDNA of both liverwort groups also with the extended E-box amplicon. Again, PCR products of expected sizes were obtained for cDNAs from jungermanniid liverworts and H. mnioides but not from marchantiid liverworts, whereas RT-PCR products were retrieved for the histone H3 positive control throughout (fig. 5). To verify the nature of the PCR products obtained for the extended amplicon, we again cloned and sequenced them. Ultimately, 167 different sequences which proved to encode bona fide PLS protein reading frames were obtained (table 1).
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Diversity of DYW-Type Genes Correlates with the Number of RNA Editing Sites
Particularly strong PCR amplification of the E-DYW amplicon was observed for the isolated liverwort Haplomitrium, which could indicate a significant degree of PPR protein diversity through extension of the gene family, in line with the high frequency of RNA editing in this taxon. Indeed, in a total of only 56 E-DYW amplicon clones, 44 different sequences could already be identified after subtracting 5 cDNA/DNA pairs (fig. 6). This diversity of DYW proteins in H. mnioides indeed appears to confirm the expectation of large DYW gene families in species with high rates of RNA editing in mitochondria. Strikingly, in the model moss Physcomitrella patens with minimally 7 RNA editing sites (Terasawa et al. 2006
) and a maximum of 49 editing sites (unpublished data) in mitochondria, only 10 DYW genes were found in the preliminary full genome sequence (http://www.cosmoss.org) by Blast similarity searches (data not shown).
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Discussion |
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The participation of PPR genes in the process of RNA editing has been discussed previously (Lurin et al. 2004
; Shikanai 2006; Salone et al. 2007). By now, 2 members of the PLS family (CRR4 and CRR21, both members of the E+ subgroup) were identified as recognition factors of specific RNA editing sites in plastids through functional genetic analyses, but none of these seem to possess a catalytic domain with a deaminase function (Kotera et al. 2005; Okuda et al. 2006, 2007). In our studies, we have focused specifically on DYW subgroup genes containing the C-terminal DYW domain with structural and distant sequence similarities to cytidine deaminases (Salone et al. 2007). As an additional hint for DYW proteins playing a role in RNA editing, a locus which affects editing of mitochondrial ccb206 transcripts was found to be near 2 DYW genes in A. thaliana (Bentolila et al. 2005). In previous phylogenetic studies, we were able to identify the C-terminal 39 amino acid segment of the DYW domain in different plants featuring organellar RNA editing to various degrees including the jungermanniid liverworts and the liverwort H. mnioides (Salone et al. 2007). This taxon in particular is now clearly corroborated in a phylogenetic position basal to the split of the 2 major clades of jungermanniid and marchantiid liverworts (Crandall-Stotler et al. 2005; Forrest and Crandall-Stotler 2005; Heinrichs et al. 2005; Groth-Malonek et al. 2007). In contrast, neither RNA editing nor DYW domains could be identified in algae (Chaetosphaeridium, Chlamydomonas, Chlorokybus, Closterium, Coleochaete, and Klebsormidium) or the marchantiid liverworts (Salone et al. 2007), the sole embryophyte clade obviously lacking RNA editing (Malek et al. 1996; Freyer et al. 1997; Steinhauser et al. 1999). However, it had remained unclear whether the detected sequences (Salone et al. 2007) are parts of complete PPR genes and furthermore whether these genes are transcribed at a detectable level in liverworts.
With the present data, we have now extended the previous results on the cDNA level and investigated the appearance of complete DYW genes in jungermanniid liverworts. We verified the conserved combination of protein domains E, E+, and DYW in 5 different jungermanniid liverworts and the basal tracheophyte I. engelmannii. Additionally, we could demonstrate the existence of a linkage between those C-terminal domains and preceding PPR motifs (typical for the extended subgroups of PLS proteins in the angiosperms) in 2 different jungermanniid species (Fossombronia cf. alaskana and D. albicans). A combination of E/E+/DYW continuity and PPR motif segments described in Rivals et al. (2006) have accordingly taken place early in land plant evolution. Hence, the DYW subgroup of PLS proteins most likely arose, just like RNA editing (Steinhauser et al. 1999), in the first embryophytes. This is further supported by the results of the transcription analyses of DYW genes in H. mnioides and in all tested jungermanniid liverwort species. In contrast, no evidence for presence of such genes could be obtained for the Marchantiidae.
PCRs with DNA or cDNA of different embryophyte species (gymnosperms, ferns, lycopodiophytes, hornworts, mosses, and liverworts) targeting the E/E+/DYW continuity resulted in diverse clones with coding sequences containing the same structure and conserved motifs as described for angiosperms (Lurin et al. 2004). In A. thaliana, O. sativa, and P. patens (3 species with completely sequenced organellar and nuclear genomes), a correlation between the numbers of DYW genes and the numbers of known RNA editing sites is suggestive (data not shown). Furthermore, the obvious diversity of DYW genes in different species correlates with the diversity of RNA editing patterns which also generally differ, even between closely related species (Schmitz-Linneweber et al. 2002; Duff 2006) and likewise with the efficiency of RNA editing which even varies in different ecotypes of the same species (Bentolila et al. 2005; Tillich et al. 2005). The conserved structure and invariant amino acids in the DYW domain on the other hand indicate a conserved role in RNA editing, possibly directly in the deamination process of cytidine to uridine. However, all investigated species feature several fold (e.g., ca., 5- to 6-fold in Arabidopsis) more RNA editing sites than DYW genes, which immediately rules out a 1:1 model of RNA editing site recognition. Hence, if the DYW proteins are indeed directly involved in the recognition of RNA editing sites and the catalyzation of the deamination process in mitochondria, individual proteins should participate in more than one editing event and there differentially interact with additional specificity factors (most likely non-DYW–type PPR proteins). This is in line with the observations that at least same trans-acting protein factors, supported by cross-competition experiments, recognize several target sites in chloroplasts (Chateigner-Boutin and Hanson 2002; Heller et al. 2008) as well as in mitochondria (Choury et al. 2004).
Our results suggest the complete lack of E/E+/DYW assemblies in 12 different marchantiid liverworts on transcript and also on genomic level. In additional support for our findings, no DYW-like sequences could be identified among the more than 33,000 different expressed sequence tags (ESTs) meantime available for Marchantia polymorpha (Salone et al. 2007). As convincing as the presence of DYW-type PPR proteins in species with known RNA editing may be, we are left with a puzzling dilemma given that the genus Haplomitrium is convincingly placed phylogenetic basal to the split of (RNA editing) jungermanniid and (non-RNA editing) marchantiid liverworts (Crandall-Stotler et al. 2005; Forrest and Crandall-Stotler 2005; Heinrichs et al. 2005; Groth-Malonek et al. 2007). Most interesting in that regard will be the inclusion of species from Treubiidae now convincingly placed as sister group to the Haplomitriidae in a class Haplomitriopsida (or Treubiopsida) by those phylogenetic studies. Unfortunately, biological material of Treubia and Apotreubia is yet more rare than of Haplomitrium, and we were so far unable to include these taxa in our studies. Hence, the absence of DYW genes and RNA editing in marchantiid liverworts is most easily explained as a secondary loss (fig. 7) and it remains enigmatic how and why a whole gene family has disappeared in that plant clade. Complete genomic sequences of marchantiid liverworts could in the (distant) future certainly reveal vestigial traces of DYW motifs as further corroboration of this hypothesis. There is at least some indication that PPR genes can indeed be subject of an accelerated evolution shaping genomes at larger scales. In several angiosperms specific PPR genes, called Rf (restorer of fertility) genes, suppress the cytoplasmic male sterility (CMS) phenotype caused by recombination events in the mitochondrial genomes (Bentolila et al. 2002; Brown et al. 2003; Desloire et al. 2003; Kazama and Toriyama 2003; Koizuka et al. 2003). Whereas most PPR genes are distributed throughout the genome in A. thaliana (Lurin et al. 2004), the Rf genes cluster together on chromosome 1 and at least some duplication events occurred only very recently on evolutionary timescales (Shikanai 2006). It might well be possible that original DYW genes initially were quickly multiplied by repeated duplication and coevolved with new RNA editing sites. Only later in evolution, they were separated by large-scale genomic rearrangements (Geddy and Brown 2007). Interestingly, all so far identified DYW genes (Lurin et al. 2004) or DYW gene parts are intronless.
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As intriguing as the here demonstrated correlation between presence of nuclear DYW genes and organellar RNA editing among embryophytes is, the functional linkage has yet to be convincingly demonstrated. One single DYW protein in A. thaliana, CRR2, is characterized so far in detail and takes part in the chloroplast pre-mRNA processing of a cotranscript of rps7 and ndhB (Hashimoto et al. 2003
). In crr2 mutants, editing of the transcript is not obviously affected, but a role in RNA editing cannot entirely ruled out as not all RNA editing sites in the mutant are catalogued and analyzed. In the light of a manageable number of DYW genes (10) and a low number of RNA editing sites (less than 50 in mitochondrial transcripts), we will use the model moss P. patens in the future to investigate a functional correlation of DYW proteins and RNA editing in mitochondria. The recently developed RIP-chip assays (RNA immunoprecipitation and chip hybridization) (Schmitz-Linneweber et al. 2005) will play a major experimental role in the approach to identify mitochondrial transcripts that coimmunoprecipitate with DYW proteins.
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Acknowledgements |
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The authors are grateful to colleagues J. Heinrichs and R. Gradstein (Goettingen), J-P. Frahm and M. Groth-Malonek (Bonn), H. Deguchi and M. Shimamura (Hiroshima), H. Muhle (Ulm), H. Becker (Saarbrücken), and I. Capesius (Heidelberg) who have helped to obtain plant materials.
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Footnotes |
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Charles Delwiche, Associate Editor
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