Géraldine Bonnard

Molecular mechanisms of cytochrome c biogenesis: three distinct systems

The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c‐type cytochromes. The hallmark of c‐type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys–Xxx–Yyy–Cys–His signature motif). From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein. In this review, common principles of assembly for all systems and the mmicular mechanisms predicted for each system are summarized. Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction. Accessory proteins of systems I and II co‐ordinate the positioning of these two substrates at the membrane surface for covalent ligation. The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates. For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

RNA editing in plant mitochondria and chloroplasts

In the mitochondria and chloroplasts of higher plants there is an RNA editing activity responsible for specific C-to-U conversions and for a few U-to-C conversions leading to RNA sequences different from the corresponding DNA sequences. RNA editing is a post-transcriptional process which essentially affects the transcripts of protein coding genes, but has also been found to modify non-coding transcribed regions, structural RNAs and intron sequences. RNA editing is essential for correct gene expression: proteins translated from edited transcripts are different from the ones deduced from the genes sequences and usually present higher similarity to the corresponding non-plant homologues. Initiation and stop codons can also be created by RNA editing. RNA editing has also been shown to be required for the stabilization of the secondary structure of introns and tRNAs.The biochemistry of RNA editing in plant organelles is still largely unknown. In mitochondria, recent experiments indicate that RNA editing may be a deamination process. A plastid transformation technique showed to be a powerful tool for the study of RNA editing. The biochemistry as well as the evolutionary features of RNA editing in both organelles are compared in order to identify common as well as organelle-specific components.

Expression of the wheat mitochondrial nad3-rps12 transcription unit: correlation between editing and mRNA maturation.

In plant mitochondria, RNA editing involves the conversion of cytidines in the genomic DNA into uridines in the corresponding RNA. Analysis of cDNAs prepared by reverse transcription of mitochondrial RNAs has shown that partially edited RNAs are present in wheat mitochondria. The extent of this partial editing as well as its potential influence on the corresponding protein sequence were studied along with the expression of a wheat mitochondrial locus. The sequence, expression, and RNA editing of the wheat mitochondrial transcription unit containing four open reading frames (nad3, rps12, orf299, orf156), all cotranscribed into a same predominant precursor RNA, have been studied. The product of orf156 is an 18-kD mitochondrial membrane protein of unknown function, whereas the product of orf299 could not be detected and this sequence seems to be a pseudogene. Sequences of cDNA clones derived by the polymerase chain reaction technique show that nad3, rps12, and orf156 transcripts are edited, whereas orf299 is not edited, except for a sequence identical to part of the coxII gene. Analysis of cDNA clones obtained from the precursor RNA shows the presence of a large number of partially edited nad3-rps12 transcripts with no evident polarity for the editing process. This shows that RNA editing is a post-transcriptional event. In addition, study of partial editing at the level of precursor, mature, and polysomal transcripts shows that mainly mature, completely edited sequences are used for translation. Deletions of a nucleotide at editing sites were observed in a number of cDNA clones, suggesting that C----U RNA editing in plant mitochondria would be achieved by nucleotide replacement.

Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity

The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified.

AtNTRB is the major mitochondrial thioredoxin reductase in Arabidopsis thaliana

NADPH‐dependent thioredoxin reductases (NTR) are homodimeric enzymes that reduce thioredoxins. Two genes encoding NADPH‐dependent thioredoxin reductases (AtNTRA and AtNTRB) were found in the genome of Arabidopsis thaliana. These originated from a recent duplication event and the encoded proteins are highly homologous. Previously, AtNTRA was shown to encode a dual targeted cytosol and mitochondrial protein. Here, we show that the AtNTRB gene encodes two mRNAs, presumably by initiating transcription at two different sites. The longer mRNA encodes a precursor polypeptide that is actively imported into mitochondria by a cleavage‐associated mechanism, while the shorter mRNA encodes a cytosolic isoform. Isolation of Arabidopsis mutants with knocked‐out AtNTRA or AtNTRB genes allowed us to prove that both genes encode cytosolic and mitochondrial isoforms. Interestingly, AtNTRB appeared to express the major mitochondrial NTR, while AtNTRA expresses as the major cytosolic isoform, suggesting that these two recently duplicated genes are evolving towards a specific function.

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

SummaryThe T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.

Biochemical requirements for the maturation of mitochondrial c-type cytochromes

Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.

Nucleotide sequence of the split tRNA UAA Leu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

SummaryThe gene encoding the tRNAUAALeufrom broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNAUAALeuintron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5′-and 3′-flanking regions of the broad bean and maize chloroplast tRNAUAALeugenes.

RNA editing in plant mitochondria.

Abstract In plant mitochondria, RNA editing involves the conversion of specific Cs in the genomic sequence into Us in the mRNA. There are a few reverse conversions. A majority of the editing sites are in coding regions. After RNA editing, plant mitochondria use the universal genetic code if one considers the RNA message. Considering the deduced protein sequence, RNA editing is a correction mechanism. RNA editing is a post‐transcriptional process, active during the maturation of the mRNA. It has no evident polarity. At the translation level, the mRNAs are likely to be fully edited and to code for a unique protein. RNA editing is a recently discovered process that may have a number of consequences on our understanding of the regulation of mitochondrial gene expression. The main problems to be solved are the identification of the template information necessary for the specificity of RNA editing and its biochemical mechanism. Comparative studies could perhaps answer the question of the evolutionary origin of ...