The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

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.

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution.

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6⌬ strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5 part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.The exoribonucleolytic activity of the exosome is fundamental to many aspects of RNA metabolism. The exosome is involved in mRNA turnover and degradation of defective and cryptic transcripts, but also in processing of the 3Ј extremities of a variety of noncoding RNAs and elimination of RISCcleaved mRNA (for recent reviews, see references 27 and 46). While most exosome functions were initially characterized in the yeast Saccharomyces cerevisiae, related complexes are present in all higher eukaryotes investigated and in several Archaea (4, 9-11, 17, 26, 29, 35, 36).Eukaryotic exosomes are composed of a core complex with which cytoplasm-and nucleus-specific subunits associate (reviewed in reference 38). One of these subunits, Rrp6p, a member of the RNase D family, associates with the nuclear exosome in yeast (2). Its human counterpart, PM/Scl-100, is predominantly nuclear but is also detected in the cytoplasm (7). Rrp6p plays a role in nuclear mRNA surveillance and in the degradation of rRNA maturation by-products or intergenic transcripts (30,33,47,51). In addition, Rrp6p is involved in the final steps in processing several noncoding RNAs (1, 6).In yeast, the TRAMP complex polyadenylates RNA substrates, which triggers their degradation by the nuclear exosome (30,47,51). In higher eukaryotes, evidence for polyadenylation of nuclear transcripts destined for degradation is emerging. Short poly(A) tails were detected upon cotranscriptional cleavage of human ␤-globin and murine serum albumin pre-mRNA (50). Human rRNA can also be polyadenylated at putative sites of endonucleolytic cleavage (44). In plants, polyadenylation of nuclear noncoding RNA also occurs, as polyadenylated transcripts of the low-abundance 5S rRNA spacer were reported in Nicotiana (20). During revision of our manuscript, a genome-wide search for exosome substrates revealed that a wide range of nuclear noncoding transcripts a...