Efficient Replication of the Plastid Genome Requires an Organellar Thymidine Kinase.

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis.

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.

The RAD52-like protein ODB1 is required for the efficient excision of two mitochondrial introns spliced via first-step hydrolysis.

Transcript splicing in plant mitochondria involves numerous nucleus-encoded factors, most of which are of eukaryotic origin. Some of these belong to protein families initially characterised to perform unrelated functions. The RAD52-like ODB1 protein has been reported to have roles in homologous recombination-dependent DNA repair in the nuclear and mitochondrial compartments in Arabidopsis thaliana. We show that it is additionally involved in splicing and facilitates the excision of two cis-spliced group II introns, nad1 intron 2 and nad2 intron 1, in Arabidopsis mitochondria. odb1 mutants lacking detectable amounts of ODB1 protein over-accumulated incompletely spliced nad1 and nad2 transcripts. The two ODB1-dependent introns were both found to splice via first-step hydrolysis and to be released as linear or circular molecules instead of lariats. Our systematic analysis of the structures of excised introns in Arabidopsis mitochondria revealed several other hydrolytically spliced group II introns in addition to nad1 intron 2 and nad2 intron 1, indicating that ODB1 is not a general determinant of the hydrolytic splicing pathway.

A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.

The plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNA-affinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria.

RecA-dependent DNA repair results in increased heteroplasmy of the Arabidopsis 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.

Plant mitochondrial genes can be expressed from mRNAs lacking stop codons.

The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT-PCR. There is no evidence that an alternative stop codon is created post-transcriptionally, either by RNA editing or by polyadenylation. The non-stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non-stop mRNAs.

Arabidopsis tRNA adenosine deaminase arginine edits the wobble nucleotide of chloroplast tRNAArg(ACG) and is essential for efficient chloroplast translation.

RNA editing changes the coding/decoding information relayed by transcripts via nucleotide insertion, deletion, or conversion. Editing of tRNA anticodons by deamination of adenine to inosine is used both by eukaryotes and prokaryotes to expand the decoding capacity of individual tRNAs. This limits the number of tRNA species required for codon-anticodon recognition. We have identified the Arabidopsis thaliana gene that codes for tRNA adenosine deaminase arginine (TADA), a chloroplast tRNA editing protein specifically required for deamination of chloroplast (cp)-tRNAArg(ACG) to cp-tRNAArg(ICG). Land plant TADAs have a C-terminal domain similar in sequence and predicted structure to prokaryotic tRNA deaminases and also have very long N-terminal extensions of unknown origin and function. Biochemical and mutant complementation studies showed that the C-terminal domain is sufficient for cognate tRNA deamination both in vitro and in planta. Disruption of TADA has profound effects on chloroplast translation efficiency, leading to reduced yields of chloroplast-encoded proteins and impaired photosynthetic function. By contrast, chloroplast transcripts accumulate to levels significantly above those of wild-type plants. Nevertheless, absence of cp-tRNAArg(ICG) is compatible with plant survival, implying that two out of three CGN codon recognition occurs in chloroplasts, though this mechanism is less efficient than wobble pairing.

Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana.

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 rrp6Delta 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 plant-specific ssDNA binding protein OSB1 is involved in the stoichiometric transmission of mitochondrial DNA in Arabidopsis.

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-beta-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.

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.

Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.

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.