Plant mitochondrial genomes exist in a natural state of heteroplasmy, in which substoichiometric levels of alternative mitochondrial DNA (mtDNA) molecules coexist with the main genome. These subgenomes either replicate autonomously or are created by infrequent recombination events. We found that Arabidopsis thaliana OSB1 (for Organellar Single-stranded DNA Binding protein1) is required for correct stoichiometric mtDNA transmission. OSB1 is part of a family of plant-specific DNA binding proteins that are characterized by a novel motif that is required for single-stranded DNA binding. The OSB1 protein is targeted to mitochondria, and promoter-b-glucuronidase fusion showed that the gene is expressed in budding lateral roots, mature pollen, and the embryo sac of unfertilized ovules. OSB1 T-DNA insertion mutants accumulate mtDNA homologous recombination products and develop phenotypes of leaf variegation and distortion. The mtDNA rearrangements occur in two steps: first, homozygous mutants accumulate subgenomic levels of homologous recombination products; second, in subsequent generations, one of the recombination products becomes predominant. After the second step, the process is no longer reversible by backcrossing. Thus, OSB1 participates in controlling the stoichiometry of alternative mtDNA forms generated by recombination. This regulation could take place in gametophytic tissues to ensure the transmission of a functional mitochondrial genome.

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.H igher plants require mitochondrial (mt) function for their survival, which depends on proper mtDNA maintenance and expression (1). At the structural level, the mtDNA of plants is relatively large, and in most species it is constantly reorganized by recombination between repeated sequences (2). Although large portions of mtDNA have been moved around during evolution, the plant mt genome evolves very slowly through nucleotide substitution; plant mt gene sequences have remained remarkably constant, suggesting the existence of very efficient DNA repair systems. On the other hand, the expression of plant mt genes is also complexly regulated, both at the transcriptional, posttranscriptional, and translational levels. For proper maturation of its transcripts, mitochondria puts to play complex processes of intron splicing, 5Ј and 3Ј RNA trimming, extensive RNA editing by C to U conversions, and regulation of transcript stability by secondary structures and polyadenylation (3, 4). Despite the importance of these mt processes in plant development, little is known about the factors involved, but at the core of the protein complexes must be DNA-binding proteins involved in mtDNA replication, recombination, repair and transcription, and RNA-binding proteins involved in posttranscriptional RNA maturation and translation. As a first step in the dissection of these complexes, we undertook to purify and identify nucleic acid-binding proteins from plant mitochondria. Most of the proteins identified are plant-specific, and many belong to a previously undescribed family of mt RNA-binding proteins (mRBP) characterized by the presence of an N-terminal RNA recognition motif (RRM). This family of plant mRBPs is ph...