Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.     

Circular mitochondrial DNA species from Oenothera with unique sequences

Summary Closed circular DNAs have been isolated from Oenothera and analysed by gel electrophoresis. A number of different size classes could be observed in the supercoiled fraction from the density gradient. The five smallest size classes of 6.3, 7.0, 8.2, 9.9, and 13.5 kilobases linear length were purified and their restriction fragment sizes compared. These five mtDNA species appear to be distinct homologous populations with no detectable large homologies between thedifferent classes. Hybridization data confirm this result; the smallest species hybridizes to itself and very weakly to some of the larger DNA molecules indicating only very little common sequence arrangement between the different circular DNAs. A restriction map of the smallest size class was constructed and confirms the circular arrangement of this DNA.Abbreviations used kb kilobases or kilobasepairs - mtDNA mitochondrial DNA - EthBr Ethidium bromide - SSC 150 mM NaCl, 15 mM sodium citrate (pH 7.0)     

Complex cis-elements determine an RNA editing site in pea mitochondria

The cis-requirements for the first editing site in the atp9 mRNA from pea mitochondria were investigated in an in vitro RNA editing system. Template RNAs deleted 5′ of −20 are edited correctly, but with decreased efficiency. Deletions between −20 and the edited nucleotide abolish editing activity. Substitution of the sequences 3′ of the editing site has little effect, which suggests that the major determinants reside upstream. Stepwise mutated RNA sequences were used as templates or competitors that divide the cis-elements into several distinct regions. In the template RNAs, mutation of the sequence between −40 and −35 reduces the editing activity, while the region from −15 to −5 is essential for the editing reaction. In competition experiments the upstream region can be titrated, while the essential sequence near the editing site is largely resistant to excess competitor. This observation suggests that either one trans-factor attaches to these separate cis-regions with different affinities or two distinct trans-factors bind to these sequences, and one of which is present in limited amounts, wheras the other one is more abundant in the lysate.     

A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant

Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2. Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1. A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the PSI-BLAST algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1.     

The mitochondrial genome of Arabidopsis is composed of both native and immigrant information

Plants contain large mitochondrial genomes, which are several times as complex as those in animals, fungi or algae. However, genome size is not correlated with information content. The mitochondrial genome (mtDNA) of Arabidopsis specifies only 58 genes in 367 kb, whereas the 184 kb mtDNA in the liverwort Marchantia polymorpha codes for 66 genes, and the 58 kb genome in the green alga Prototheca wickerhamii encodes 63 genes. In Arabidopsis’ mtDNA, genes for subunits of complex II, for several ribosomal proteins and for 16 tRNAs are missing, some of which have been transferred recently to the nuclear genome. Numerous integrated fragments originate from alien genomes, including 16 sequence stretches of plastid origin, 41 fragments of nuclear (retro)transposons and two fragments of fungal viruses. These immigrant sequences suggest that the large size of plant mitochondrial genomes is caused by secondary expansion as a result of integration and propagation, and is thus a derived trait established during the evolution of land plants.     

Phenotypes of Alb3p and carotenoid synthesis mutants show similarities regarding light sensitivity, thylakoid structure and protein stability

Chloroplast proteins of the Alb3/YidC/Oxa1p family are necessary for assembly of photosynthetic complexes in the thylakoid membranes. Alb3p in Arabidopsis thaliana is essential for posttranslational LHCII-integration into thylakoid membranes and participates in cotranslational assembly of D1. However, the pleiotropic defects of an Alb3p mutant, albino3, suggest additional functions for Alb3p. To obtain an impression of such potential further Alb3p activities from phenotypic manifestations, properties of mutants disturbed in thylakoid membrane protein transport or carotenoid biosynthesis were compared with the albino3 mutant. Specific defects observed in albino3 were similar to those in a carotenoid synthesis mutant. While this correlation did not provide tangible evidence for Alb3p being involved in the integration of carotenoids in photosynthetic complexes, it suggests a possible avenue for future investigations.     

Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

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.