A hypothesis on the identification of the editing enzyme in plant organelles

RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica

The plant‐specific pentatricopeptide repeat (PPR) proteins with variable PPR repeat lengths (PLS‐type) and protein extensions up to the carboxyterminal DYW domain have received attention as specific recognition factors for the C‐to‐U type of RNA editing events in plant organelles. Here, we report a DYW‐protein knockout in the model plant Physcomitrella patens specifically affecting mitochondrial RNA editing positions cox1eU755SL and rps14eU137SL. Assignment of DYW proteins and RNA editing sites might best be corroborated by data from a taxon with a slightly different, yet similarly manageable low number of editing sites and DYW proteins. To this end we investigated the mitochondrial editing status of the related funariid moss Funaria hygrometrica. We find that: (i) Funaria lacks three mitochondrial RNA editing positions present in Physcomitrella, (ii) that F. hygrometrica cDNA sequence data identify nine DYW proteins as clear orthologues of their P. patens counterparts, and (iii) that the ‘missing’ 10th DYW protein in F. hygrometrica is responsible for two mitochondrial editing sites in P. patens lacking in F. hygrometrica (nad3eU230SL, nad4eU272SL). Interestingly, the third site of RNA editing missing in F. hygrometrica (rps14eU137SL) is addressed by the DYW protein characterized here and the presence of its orthologue in F. hygrometrica is explained through its simultaneous action on site cox1eU755SL conserved in both mosses.     

Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

Introducing Intron Locus cox1i624 for Phylogenetic Analyses in Bryophytes: On the Issue of Takakia as Sister Genus to All Other Extant Mosses

Liverworts are well supported as the sister group to all other land plants (embryophytes) by molecular data. Observations strongly supporting this earliest dichotomy in embryophyte evolution are the strikingly different introns occurring in the mitochondrial DNAs of liverworts versus non-liverwort embryophytes (NLE), including the mosses. A final conclusion on the most basal lineages of mosses, for which genera such as Sphagnum and Takakia are the most likely candidates, is lacking. We have now investigated cox1i624, a mitochondrial group I intron conserved between the moss Physcomitrella patens and the liverwort Marchantia polymorpha. Focusing on a sampling of liverwort and moss genera, which had previously been identified as early branching taxa in their respective clades, we find that group I intron cox1i624 is universally conserved in all 33 mosses and 11 liverworts investigated. The group I intron core secondary structure is well conserved between the two ancient land plant clades. However, whereas dramatic size reductions are seen in the moss phylogeny, exactly the opposite is observed for liverworts. The cox1i624g1 locus was used for phylogenetic tree reconstruction also in combination with data sets of nad5i753g1 as well as chloroplast loci rbcL and rps4. The phylogenetic analyses revealed (i) very good support for the Treubiopsida as sister clade to all other liverworts, (ii) a sister group relationship of the nematodontous Tetraphidopsida and Polytrichopsida and (iii) two rivalling hypotheses about the basal-most moss genus with mitochondrial loci suggesting an isolated Takakia as sister to all other mosses and chloroplast loci indicating a Takakia–Sphagnum clade.     

DYW-type PPR proteins in a heterolobosean protist: plant RNA editing factors involved in an ancient horizontal gene transfer?

A particular type of pentatricopeptide repeat (PPR) proteins with variable length of the 35 aa PPR motifs and conserved carboxyterminal extensions, named the PLS proteins, was so far exclusively identified in land plants. Several PLS proteins with such domain extensions (E, E+, DYW) were shown to be involved in plant organellar RNA editing but their evolutionary origin had remained enigmatic. We here report the first case of DYW-type PLS proteins outside of the plant kingdom in the protist Naegleria gruberi and hypothesize on horizontal gene transfer in very early land plant evolution.     

Plant Mitochondrial RNA Editing

RNA editing affects messenger RNAs and transfer RNAs in plant mitochondria by site-specific exchange of cytidine and uridine bases in both seed and nonseed plants. Distribution of the phenomenon among bryophytes has been unclear since RNA editing has been detected in some but not all liverworts and mosses. A more detailed understanding of RNA editing in plants required extended data sets for taxa and sequences investigated. Toward this aim an internal region of the mitochondrial nad5 gene (1104 nt) was analyzed in a large collection of bryophytes and green algae (Charales). The genomic nad5 sequences predict editing in 30 mosses, 2 hornworts, and 7 simple thalloid and leafy liverworts (Jungermanniidae). No editing is, however, required in seven species of the complex thalloid liverworts (Marchantiidae) and the algae. RNA editing among the Jungermanniidae, on the other hand, reaches frequencies of up to 6% of codons being modified. Predictability of RNA editing from the genomic sequences was confirmed by cDNA analysis in the mosses Schistostega pennata and Rhodobryum roseum, the hornworts Anthoceros husnotii and A. punctatus, and the liverworts Metzgeria conjugata and Moerckia flotoviana. All C-to-U nucleotide exchanges predicted to reestablish conserved codons were confirmed. Editing in the hornworts includes the removal of genomic stop codons by frequent reverse U-to-C edits. Expectedly, no RNA editing events were identified by cDNA analysis in the marchantiid liverworts Ricciocarpos natans, Corsinia coriandra, and Lunularia cruciata. The findings are discussed in relation to models on the phylogeny of land plants. Received: 2 April 1998 / Accepted: 4 August 1998     

The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain

In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.     

The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana

RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.     

The mitochondrial genomes of the early land plants Treubia lacunosa and Anomodon rugelii: dynamic and conservative evolution

Early land plant mitochondrial genomes captured important changes of mitochondrial genome evolution when plants colonized land. The chondromes of seed plants show several derived characteristics, e.g., large genome size variation, rapid intra-genomic rearrangement, abundant introns, and highly variable levels of RNA editing. On the other hand, the chondromes of charophytic algae are still largely ancestral in these aspects, resembling those of early eukaryotes. When the transition happened has been a long-standing question in studies of mitochondrial genome evolution. Here we report complete mitochondrial genome sequences from an early-diverging liverwort, Treubia lacunosa, and a late-evolving moss, Anomodon rugelii. The two genomes, 151,983 and 104,239 base pairs in size respectively, contain standard sets of protein coding genes for respiration and protein synthesis, as well as nearly full sets of rRNA and tRNA genes found in the chondromes of the liverworts Marchantia polymorpha and Pleurozia purpurea and the moss Physcomitrella patens. The gene orders of these two chondromes are identical to those of the other liverworts and moss. Their intron contents, with all cis-spliced group I or group II introns, are also similar to those in the previously sequenced liverwort and moss chondromes. These five chondromes plus the two from the hornworts Phaeoceros laevis and Megaceros aenigmaticus for the first time allowed comprehensive comparative analyses of structure and organization of mitochondrial genomes both within and across the three major lineages of bryophytes. These analyses led to the conclusion that the mitochondrial genome experienced dynamic evolution in genome size, gene content, intron acquisition, gene order, and RNA editing during the origins of land plants and their major clades. However, evolution of this organellar genome has remained rather conservative since the origin and initial radiation of early land plants, except within vascular plants.     

A conserved DYW domain of the pentatricopeptide repeat protein possesses a novel endoribonuclease activity

Many plant pentatricopeptide repeat (PPR) proteins are known to contain a highly conserved C-terminal DYW domain whose function is unknown. Recently, the DYW domain has been proposed to play a role in RNA editing in plant organelles. To address this possibility, we prepared recombinant DYW proteins and tested their cytidine deaminase activity. However, we could not detect any activity in the assays we used. Instead, we found that the recombinant DYW domains possessed endoribonuclease activity and cleaved before adenosine residues in the RNA molecule. Some DYW-containing PPR proteins may catalyze site-specific cleavage of target RNA species.     

Exclusive conservation of mitochondrial group II intron nad4i548 among liverworts and its use for phylogenetic studies in this ancient plant clade

Liverworts occupy a pivotal position in land plant (embryophyte) phylogeny as the presumed earliest-branching major clade, sister to all other land plants, including the mosses, hornworts, lycophytes, monilophytes and seed plants. Molecular support for this earliest dichotomy in land plant phylogeny comes from strikingly different occurrences of introns in mitochondrial genes distinguishing liverworts from all other embryophytes. Exceptionally, however, the nad5 gene--the mitochondrial locus hitherto used most widely to elucidate early land plant phylogeny--carries a group I type intron that is shared between liverworts and mosses. We here explored whether a group II intron, the other major type of organellar intron, would similarly be conserved in position across the entire diversity of extant liverworts and could be of use for phylogenetic analyses in this supposedly most ancient embryophyte clade. To this end, we investigated the nad4 gene as a candidate locus possibly featuring different introns in liverworts as opposed to the non-liverwort embryophyte (NLE) lineage. We indeed found group II intron nad4i548 universally conserved in a wide phylogenetic sampling of 55 liverwort taxa, confirming clade specificity and surprising evolutionary stability of plant mitochondrial introns. As expected, intron nad4i548g2 carries phylogenetic information in its variable sequences, which confirms and extends previous cladistic insights on liverwort evolution. We integrate the new nad4 data with those of the previously established mitochondrial nad5 and the chloroplast rbcL and rps4 genes and present a phylogeny based on the fused datasets. Notably, the phylogenetic analyses suggest a reconsideration of previous phylogenetic and taxonomic assignments for the genera Calycularia and Mylia and resolve a sister group relationship of Ptilidiales and Porellales.