We have determined the complete sequence of the mitochondrial DNA in the model plant species Arabidopsis thaliana, affording access to the first of its three genomes. The 366,924 nucleotides code for 57 identified genes, which cover only 10% of the genome. Introns in these genes add about 8%, open reading frames larger than 100 amino acids represent 10% of the genome, duplications account for 7%, remnants of retrotransposons of nuclear origin contribute 4% and integrated plastid sequences amount to 1%-leaving 60% of the genome unaccounted for. With the significant contribution of duplications, imported foreign DNA and the extensive background of apparently functionless sequences, the mosaic structure of the Arabidopsis thaliana mitochondrial genome features many aspects of size-relaxed nuclear genomes.

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Fig. 2. The MORF family of proteins contains nine genes and a potential pseudogene in A. thaliana. (A) The cladogram of similarities between the MORF proteins shows that the plastid editing factors MORF2 and MORF9 are rather distant from each other and more similar to the mitochondrial proteins MORF3 and MORF1, respectively. Predictions (marked mt or cp) and experimental data obtained by GFP-fusion protein localization (only MORF2) or proteomics MS data (marked with an asterisk) for the respective organellar locations are indicated. The MORF8 protein encoded by At3g15000 has been found in mitochondria in three independent assays. Proteins investigated here for their function are boxed. The conserved ∼100-amino acids domain is shaded; the other sequences show much less conservation (SI Appendix, Fig. S2). The potential pseudogene (At1g53260) contains only the C-terminal part of this conserved region. (B) Exon structures of the MORF3, MORF4 and MORF6 genes are similar to the MORF1 locus and contribute similar fragments but differ in their C-terminal extensions. MORF3 is a mitochondrial editing factor involved at more than 40 sites. Locations of the T-DNA insertions in mutants morf3-1, morf4-1, and morf6-1 are shown. LB denotes the location of the left border of the T-DNA. (C) Numbers of editing sites affected by T-DNA insertions in the respective MORF genes. In mutants morf4-1 and morf6-1, only one noncoding site each shows somewhat reduced editing.

RNA editing alters the identity of nucleotides in RNA molecules such that the information for a protein in the mRNA differs from the prediction of the genomic DNA. In chloroplasts and mitochondria of flowering plants, RNA editing changes C nucleotides to U nucleotides; in ferns and mosses, it also changes U to C. The approximately 500 editing sites in mitochondria and 40 editing sites in plastids of flowering plants are individually addressed by specific proteins, genes for which are amplified in plant species with organellar RNA editing. These proteins contain repeat elements that bind to cognate RNA sequence motifs just 5' to the edited nucleotide. In flowering plants, the site-specific proteins interact selectively with individual members of a different, smaller family of proteins. These latter proteins may be connectors between the site-specific proteins and the as yet unknown deaminating enzymatic activity.

Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L (long) and L2 (long2) and the S2 (short2) motifs. We now find that inclusion of these motifs improves the prediction of RNA editing target sites. Previously overlooked RNA editing target sites are suggested from the PPR motif structures of known E-class PPR proteins and are experimentally verified. RNA editing target sites are assigned for the novel PPR protein MEF32 (mitochondrial editing factor 32) and are confirmed in the cDNA.

Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.Plant and fungal mitochondria have highly branched electron transport chains. The protonpumping respiratory complexes I, III, and IV work to a varying extent in parallel with non-protonpumping enzymes, i.e. type II NAD(P)H dehydrogenases and alternative oxidase. Thus, the coupling of electron transport to ATP formation varies depending on the electron path (Siedow and Umbach, 1995;Joseph-Horne et al., 2001).Complex I, the proton-pumping (type I) NAD(P)H dehydrogenase, is a multisubunit enzyme that is inhibited by rotenone in most organisms. It is present in ␣-proteobacteria and mitochondria of all eukaryotes except fermenting yeasts e.g. Saccharomyces cerevisiae. A homologous complex with unclear enzymatic properties is also present in chloroplasts (Yagi, 1991;Friedrich et al., 1995;Rasmusson et al., 1998).Type II, or rotenone-insensitive, NAD(P)H dehydrogenases have been found in several bacterial species and in plant and fungal mitochondria. The most studied enzymes, Escherichia coli NDH and S. cerevisiae NDI1, are FAD-containing single-polypeptide enzymes of 45 to 50 kD de Vries and Grivell, 1988). The S. cerevisiae NDI1 is located on the inner surface of the inner mitochondrial membrane, where it catalyzes the oxidation of matrix NADH (de Vries et al., 1992). Two homologous S. cerevisiae proteins, NDE1 and NDE2, are located on the external side of the inner membrane, where they oxidize cytoplasmic NADH (Luttik et al., 1998;Small and McAlister-Henn, 1998). An NADPHspecific type II dehydrogenase, NDE1, is present on the external surface of the inner membrane of Neurospora crassa ...

RNA editing in flowering plant mitochondria alters 400 to 500 nucleotides from C to U, changing the information content of most mRNAs and some tRNAs. So far, none of the specific or general factors responsible for RNA editing in plant mitochondria have been identified. Here, we characterize a nuclear-encoded gene that is involved in RNA editing of three specific sites in different mitochondrial mRNAs in Arabidopsis thaliana, editing sites rps4-956, nad7-963, and nad2-1160. The encoded protein MITOCHONDRIAL RNA EDITING FACTOR1 (MEF1) belongs to the DYW subfamily of pentatricopeptide repeat proteins. Amino acid identities altered in MEF1 from ecotype C24, in comparison to Columbia, lower the activity at these editing sites; single amino acid changes in mutant plants inactivate RNA editing. These variations most likely modify the affinity of the editing factor to the affected editing sites in C24 and in the mutant plants. Since lowered and even absent RNA editing is tolerated at these sites, the amino acid changes may be silent for the respective protein functions. Possibly more than these three identified editing sites are addressed by this first factor identified for RNA editing in plant mitochondria.