Patrice Imbault

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–β-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.

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.

Characterization of trans-splicing in Euglenoids.

Abstract We have looked for trans-splicing of nuclear mRNAs in several Euglenoid species. In Cyclidiopsis acus, Phacus curvicauda, Rhabdomonas costata and Menoidium pellucidum we showed that several pre-mRNAs chosen at random are matured by a trans-splicing process: we identified SL-RNA genes whose 5′ ends (SLs for spliced leader-sequences) were transferred to the 5′ extremities of mRNAs. The SL-RNA genes are located on repeated DNA fragments which also encode 5S rRNA in P. curvicauda and C. acus. The potential secondary structures of SL-RNAs are compared to those previously characterized in two other Euglenoids: Euglena gracilis and Entosiphon sulcatum. In another Euglenoid species, Distigma proteus, since none of the mRNAs examined were trans-spliced, it is possible that trans-splicing does not occur. Phylogeny based on 5S rRNA sequences suggests that the species which have, or have had, chloroplasts (E. gracilis, P. curvicauda, C. acus) diverged early from the others.

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.

Trans-splicing and cis-splicing in the colourless Euglenoid, Entosiphon sulcatum

Abstract The colourless Euglenoid Entosiphon sulcatum is thought to have diverged before the symbiotic event which gave rise to the photosynthetic Euglenoid species as Euglena gracilis. We have isolated genes encoding spliced leader-sequence RNA (SL-RNA) and we show that pre-mRNAs are matured via a trans-splicing reaction in E. sulcatum, as in the case of E. gracilis. The 2.5-kb repeated DNA fragment which encodes the SL-RNA gene also encodes a 5S rRNA gene as well as the genes for the small nuclear (sn) RNAs U1, U2 and U5. The presence of snRNA U1 indicates that the classical cis-splicing mechanism also exists in E. sulcatum. In addition, we show that the E. sulcatum β-tubulin gene has the intron borders GU-AG, typical of spliceosome-matured introns which have not yet been found in E. gracilis. The probable origins of trans- and cis-mechanisms in Euglenoids are discussed.

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.

Post-transcriptional regulation by light of the biosynthesis ofEuglena ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit

InEuglena gracilis, the amounts of the mature small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) increase during cell greening, while an analysis of the transcripts, performed at different stages of chloroplast development, shows no difference in the amounts of the corresponding mRNA. Pulse-chase experiments followed by immunoprecipitation show a significant increase in the rate of synthesis of the large molecular weight precursor (which consists of a transit peptide followed by eight small subunits) beginning after 12 h of illumination. Nevertheless, its half life does not change significantly during the chloroplast development. The results presented strongly suggest that the regulation of the expression of the Rubisco small subunit occurs at the translational level.

Immunological evidence for structural differences between Euglena gracilis chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic counterparts

In a plant cell, there are at least two aminoacyltRNA synthetases (EC 6.1.1. . . .) specific for the same amino acid, one in the cytoplasm and the other in the chloroplasts. There is indirect evidence that both enzymes are coded for by the nuclear genome in spite of the existence of a protein synthesis machinery inside the chloroplast (for a general review, see [ 11). The chloroplastic aminoacyl-tRNA synthetases are known to differ from their cytoplasmic counterparts in their substrate (tRNA) specificity and their chromatographic mobility [ 1 ] but so far little was known about their structural similarities or differences. 2.1. Enzymes Chloroplastic and cytoplasmic ValRS and LeuRS were purified from green cells of Euglena gracilis Z as described in [2-51. One enzyme unit is defined as the amount of enzyme which catalyzes the aminoacylation of 1 nmol of tRNAin 1 min at 30°C.

Purification of Euglena gracilis cytoplasmic leucyl tRNA synthetase

Abstract The cytoplasmic leucyl-tRNA synthetase from Euglena gracilis has been purified to homogeneity. The purification procedure includes chromatography on hydroxyapatite and on phosphocellulose, followed by Sepharose-Blue Dextran affinity chromatography and allows a 1030-fold purification of the enzyme with a recovery of 19% of the initial activity. The purified leucyl-tRNA synthetase (LeuRS) is a monomer of Mr 116 000. The affinity constants of the enzyme are 2.4 · 10−5 M for L-leucine, 1.1 · 10−5 M for ATP, 1.6 · 10−6 for Euglena tRNALeu and 2 · 10−7 M for yeast tRNA3Leu.

Chloroplastic and cytoplasmic valyl- and leucyl- tRNA synthetases from Euglena gracilis: Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.