Nuclear DYW-type PPR gene families diversify with increasing RNA editing frequencies in liverwort and moss mitochondria.

RNA editing in mitochondria and chloroplasts of land plants alters transcript sequences by site-specific conversions of cytidines into uridines. RNA editing frequencies vary extremely between land plant clades, ranging from zero in some liverworts to more than 2,000 sites in lycophytes. Unique pentatricopeptide repeat (PPR) proteins with variable domain extension (E/E+/DYW) have recently been identified as specific editing site recognition factors in model plants. The distinctive functions of these PPR protein domain additions have remained unclear, although deaminase function has been proposed for the DYW domain. To shed light on diversity of RNA editing and DYW proteins at the origin of land plant evolution, we investigated editing patterns of the mitochondrial nad5, nad4, and nad2 genes in a wide sampling of more than 100 liverworts and mosses using the recently developed PREPACT program (www.prepact.de) and exemplarily confirmed predicted RNA editing sites in selected taxa. Extreme variability in RNA editing frequency is seen both in liverworts and mosses. Only few editings exist in the liverwort Lejeunea cavifolia or the moss Pogonatum urnigerum whereas up to 20% of cytidines are edited in the liverwort Haplomitrium mnioides or the moss Takakia lepidozioides. Interestingly, the latter are taxa that branch very early within their respective clades. Amplicons targeting the E/E+/DYW domains and subsequent random clone sequencing show DYW domains among bryophytes to be highly conserved in comparison with their angiosperm counterparts and to correlate well with RNA editing frequencies regarding their diversities. We propose that DYW proteins are the key players of RNA editing at the origin of land plants.

Tracing plant Mitochondrial DNA evolution: rearrangements of the ancient mitochondrial gene cluster trnA-trnT-nad7 in liverwort phylogeny.

Whereas frequent recombination characterizes flowering plant mitochondrial genomes, some mitochondrial gene arrangements may, in contrast, be conserved between streptophyte algae and early land plant clades (bryophytes). Here we explore the evolutionary fate of the mitochondrial gene arrangement trnA-trnT-nad7, which is conserved among the alga Chara, the moss Physcomitrella, and the liverwort Marchantia, although trnT is inverted in orientation in the latter. Surprisingly, we now find that the Chara-type gene arrangement is generally conserved in mosses, but that trnT is lacking between trnA and nad7 in all simple-thalloid and leafy (jungermanniid) liverworts. The ancient gene continuity trnA-trnT-nad7 is, however, conserved in Blasia, representing the sister lineage to all other complex-thalloid (marchantiid) liverworts. The recombinogenic insertion of short sequence stretches, including nad5 and rps7 pseudogene fragments copied from elsewhere in the liverwort mtDNA, likely mediated a subsequent inversion of trnT and flanking sequences in a basal grade of marchantiid liverworts, which was then followed by an independent secondary loss of trnT in derived marchantiid taxa later in evolution. In contrast to the previously observed extreme degree of coding sequence conservation and the assumed absence of active recombination in Marchantia mtDNA, this now reveals a surprisingly dynamic evolution of marchantiid liverwort mitochondrial genomes.

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.

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae.

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.

The deepest divergences in land plants inferred from phylogenomic evidence.

Phylogenetic relationships among the four major lineages of land plants (liverworts, mosses, hornworts, and vascular plants) remain vigorously contested; their resolution is essential to our understanding of the origin and early evolution of land plants. We analyzed three different complementary data sets: a multigene supermatrix, a genomic structural character matrix, and a chloroplast genome sequence matrix, using maximum likelihood, maximum parsimony, and compatibility methods. Analyses of all three data sets strongly supported liverworts as the sister to all other land plants, and analyses of the multigene and chloroplast genome matrices provided moderate to strong support for hornworts as the sister to vascular plants. These results highlight the important roles of liverworts and hornworts in two major events of plant evolution: the water-to-land transition and the change from a haploid gametophyte generation-dominant life cycle in bryophytes to a diploid sporophyte generation-dominant life cycle in vascular plants. This study also demonstrates the importance of using a multifaceted approach to resolve difficult nodes in the tree of life. In particular, it is shown here that densely sampled taxon trees built with multiple genes provide an indispensable test of taxon-sparse trees inferred from genome sequences.

Transport of magnesium and other divalent cations: evolution of the 2-TM-GxN proteins in the MIT superfamily.

In bacteria, magnesium uptake is mainly mediated by the well-characterized CorA type of membrane proteins. In recent years, functional homologues have been characterized in the inner mitochondrial membrane of yeast and mammals (the MRS2/LPE10 type), in the plasma membrane of yeast (the ALR/MNR type) and, as an extended family of proteins, in the model plant Arabidopsis thaliana. Despite generally low sequence similarity, individual proteins can functionally complement each other over large phylogenetic distances. All these proteins are characterized by a universally conserved Gly-Met-Asn (GMN) motif at the end of the first of two conserved transmembrane domains near the C-terminus. Mutations of the GMN motif are known to abolish Mg(2+) transport, but the naturally occurring variants GVN and GIN may be associated with the transport of other divalent cations, such as zinc and cadmium, respectively. We refer to this whole class of proteins as the 2-TM-GxN type. The functional membrane channel is thought to be formed by oligomers containing four or five subunits. The wealth of sequence data now available allows us to explore the evolutionary diversification of the basic 2-TM-GxN model within the so-called metal ion transporter (MIT) superfamily. Here we report phylogenetic analyses on more than 360 homologous protein sequences derived from genomic sequences from representatives of all three domains of life. Independent gene duplications have occurred in fungi, plants and proteobacteria at different phylogenetic depths. Moreover, there is ample evidence for several instances of horizontal gene transfer of members of the 2-TM-GxN superfamily in Eubacteria and Archaea. Only single genes of the MRS2 type have been identified in vertebrate genomes. In contrast, 15 members are found in the model plant Arabidopsis thaliana, which appear to have arisen by at least four independent founder events before the diversification of flowering plants. Phylogenetic clade assignment seems to correlate with alterations in the highly conserved sequence around the GMN motif. This presumably forms an integral part of the pore surface, and changes in its structure may result in altered transport capacities for different divalent cations.

Ancestors of trans-splicing mitochondrial introns support serial sister group relationships of hornworts and mosses with vascular plants.

Some group II introns in the organelle genomes of plants and algae are disrupted and require trans-splicing of the affected exons from independent transcripts. A peculiar mitochondrial nad5 gene structure is universally conserved in flowering plants where two trans-splicing introns frame a tiny exon of only 22 nucleotides, and two additional conventional group II introns interrupt the nad5 reading frame at other sites. These four introns are absent in the liverwort Marchantia polymorpha, which carries a group I intron at an unrelated site in nad5. To determine how intron gains and losses have sculptured mitochondrial gene structures in early land-plant evolution, we have investigated the full nad5 gene structures in the three bryophyte classes and the fern Asplenium nidus. We find the single Marchantia group I intron nad5i753 present as the only intervening sequence in both closely (Corsinia and Monoclea) and distantly related (Noteroclada, Bazzania, and Haplomitrium) liverwort genera. In a taxonomically wide spectrum of mosses (Sphagnum, Encalypta, Timmia, Ulota, and Rhacocarpus); however, we additionally identify the angiosperm-type group II introns nad5i230 and nad5i1455. The latter is a cis-arranged homolog to one of the two angiosperm trans-splicing introns, notably the first of its kind in mosses. In the hornwort Anthoceros, the "moss and liverwort-type" group I intron nad5i753 is absent, and, besides nad5i230 and nad5i1455, intron nad5i1477 is present as the second ancestral group II intron which has evolved into a trans-splicing arrangement in angiosperms. The influence of highly frequent RNA editing, most notably in the genera Haplomitrium, Anthoceros, and Asplenium, on phylogenetic tree construction is investigated and discussed. Taken together, the data (1) support a sister group relationship of liverworts as a whole to all other embryophytes, (2) indicate loss of a group I and serial entries of group II introns in the nad5 gene during early evolution of the nonliverwort lineage, and (3) propose a placement of hornworts as sister group to tracheophytes.