Volker Knoop

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.

The mitochondrial DNA of land plants: peculiarities in phylogenetic perspective

Land plants exhibit a significant evolutionary plasticity in their mitochondrial DNA (mtDNA), which contrasts with the more conservative evolution of their chloroplast genomes. Frequent genomic rearrangements, the incorporation of foreign DNA from the nuclear and chloroplast genomes, an ongoing transfer of genes to the nucleus in recent evolutionary times and the disruption of gene continuity in introns or exons are the hallmarks of plant mtDNA, at least in flowering plants. Peculiarities of gene expression, most notably RNA editing and trans-splicing, are significantly more pronounced in land plant mitochondria than in chloroplasts. At the same time, mtDNA is generally the most slowly evolving of the three plant cell genomes on the sequence level, with unique exceptions in only some plant lineages. The slow sequence evolution and a variable occurrence of introns in plant mtDNA provide an attractive reservoir of phylogenetic information to trace the phylogeny of older land plant clades, which is as yet not fully resolved. This review attempts to summarize the unique aspects of land plant mitochondrial evolution from a phylogenetic perspective.

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.

The mitochondrial genome on its way to the nucleus: different stages of gene transfer in higher plants

The vast majority of mitochondrial proteins are in all eukaryotes encoded in the nuclear genomes by genes which have been transferred from the original endosymbiont. DNA as well as RNA was and is exchanged between organelies. A functionally successful information transfer, however, requires complex structural and regulatory alterations of the concerned gene. The recently identified variations of the information content in mitochondrial genomes of different plant species represent different stages of the transfer process. These evolutionary intermediates allow a definition of requirements and chances of successful gene transfers.

Plant Mitochondrial RNA Editing

Abstract. 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.

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 Mg2+ 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.

A molecular phylogeny of bryophytes based on nucleotide sequences of the mitochondrialnad5 gene

In contrast to animals, the slowly evolving mitochondrial nucleotide sequences of plants appear well suited to investigate phylogenetic relations between old taxonomic groups. Analysis ofnad5 gene sequences in 47 bryophytes, the living representatives of very early land plants, confirm this assessment. Statistically reliable phylogenetic trees are obtained with different mathematical approaches. A group I intron sequence conserved in thenad5 gene of all 30 mosses and 15 liverworts investigated supports a sister group relationship of the two classes. The intron sequence adds phylogenetic information for fine resolution on top of the conserved exon sequences down to the level of classically defined orders or families, respectively. This intron is not present in the hornwortsAnthoceros husnotii andA. punctatus. The results allow statements on diverging taxonomic interpretations and support the monophyly of the liverworts, mosses, Jungermanniidae, Marchantiidae and Bryidae, and allow recognition of subclasses like Hypnanae and Dicrananae. Among the mosses, the derived orders (subclass Bryidae) are confidently set apart from the Sphagnales, Andreaeales, Polytrichales and Tetraphidales with Buxbaumiales occupying a mediating position. Among the liverworts, full support is found for the classic separation of simple (jungermanniid) and complex thalloid (marchantiid) species with a strikingly low mitochondrial sequence divergence among the latter.

Extreme RNA Editing in Coding Islands and Abundant Microsatellites in Repeat Sequences of Selaginella moellendorffii Mitochondria: The Root of Frequent Plant mtDNA Recombination in Early Tracheophytes

Abstract Using an independent fosmid cloning approach and comprehensive transcriptome analysis to complement data from the Selaginella moellendorffii genome project, we determined the complete mitochondrial genome structure of this spikemoss. Numerous recombination events mediated mainly via long sequence repeats extending up to 7kbp result in a complex mtDNA network structure. Peculiar features associated with the repeat sequences are more than 80 different microsatellite sites (predominantly trinucleotide motifs). The S. moellendorffii mtDNA encodes a plant-typical core set of a twin-arginine translocase (tatC), 17 respiratory chain subunits, and 2 rRNAs but lacks atp4 and any tRNA genes. As a further novelty among plant chondromes, the nad4L gene is encoded within an intron of the nad1 gene. A total of 37 introns occupying the 20 mitochondrial genes (four of which are disrupted into trans-splicing arrangements including two novel instances of trans-splicing introns) make the S. moellendorffii chondrome the intron-richest and gene-poorest plant mtDNA known. Our parallel transcriptome analyses demonstrates functional splicing of all 37 introns and reveals a new record amount of plant organelle RNA editing with a total of 2,139 sites in mRNAs and 13 sites in the two rRNAs, all of which are exclusively of the C-to-U type.

A Root-Expressed Magnesium Transporter of the MRS2/MGT Gene Family in Arabidopsis thaliana Allows for Growth in Low-Mg2+ Environments

The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg2+ uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg2+ concentrations were lowered to 50 μM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.

RNA editing: only eleven sites are present in the Physcomitrella patens mitochondrial transcriptome and a universal nomenclature proposal

RNA editing in mitochondria and chloroplasts of land plants alters the coding content of transcripts through site-specific exchanges of cytidines into uridines and vice versa. The abundance of RNA editing in model plant species such as rice or Arabidopsis with some 500 affected sites in their organelle transcripts hinders straightforward approaches to elucidate its mechanisms. The moss Physcomitrella patens is increasingly being appreciated as an alternative plant model system, enhanced by the recent availability of its complete chloroplast, mitochondrial, and nuclear genome sequences. We here report the transcriptomic analysis of Physcomitrella mitochondrial mRNAs as a prerequisite for future studies of mitochondrial RNA editing in this moss. We find a strikingly low frequency of RNA editing affecting only eleven, albeit highly important, sites of C-to-U nucleotide modification in only nine mitochondrial genes. Partial editing was seen for two of these sites but no evidence for any silent editing sites (leaving the identity of the encoded amino acid unchanged) as commonly observed in vascular plants was found in Physcomitrella, indicating a compact and efficient organization of the editing machinery. Furthermore, we here wish to propose a unifying nomenclature to clearly identify and designate RNA editing positions and to facilitate future communication and database annotation.