Structural and content diversity of mitochondrial genome in beet: a comparative genomic analysis.

Despite their monophyletic origin, mitochondrial (mt) genomes of plants and animals have developed contrasted evolutionary paths over time. Animal mt genomes are generally small, compact, and exhibit high mutation rates, whereas plant mt genomes exhibit low mutation rates, little compactness, larger sizes, and highly rearranged structures. We present the (nearly) whole sequences of five new mt genomes in the Beta genus: four from Beta vulgaris and one from B. macrocarpa, a sister species belonging to the same Beta section. We pooled our results with two previously sequenced genomes of B. vulgaris and studied genome diversity at the species level with an emphasis on cytoplasmic male-sterilizing (CMS) genomes. We showed that, contrary to what was previously assumed, all three CMS genomes belong to a single sterile lineage. In addition, the CMSs seem to have undergone an acceleration of the rates of substitution and rearrangement. This study suggests that male sterility emergence might have been favored by faster rates of evolution, unless CMS itself caused faster evolution.

The rpl5- rps14 mitochondrial region: a hot spot for DNA rearrangements in Solanum spp. somatic hybrids.

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5- rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5- rps14 arrangements shown after somatic hybridization. Variability in the rpl5- rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.

Plant mitochondrial polyadenylated mRNAs are degraded by a 3'- to 5'-exoribonuclease activity, which proceeds unimpeded by stable secondary structures.

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3'-extremities. We show that a 3'- to 5'-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20-30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3' to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3' ends of mRNAs promote an efficient 3'- to 5'- degradation process.

The chloroplast-derived trnW and trnM-e genes are not expressed in Arabidopsis mitochondria.

Depending on their genetic origin, plant mitochondrial tRNAs are classified into three categories: the "native" and "chloroplast-like" mitochondrial-encoded tRNAs and the imported nuclear-encoded tRNAs. The number and identity of tRNAs in each category change from one plant specie to another. As some plant mitochondrial trn genes were found to be not expressed, and as all Arabidopsis thaliana mitochondrial trn genes are known, we systematically tested the expression of A. thaliana mitochondrial trn genes. Both the "chloroplast-like" trnW and trnM-e genes were found to be not expressed. These exceptions are remarkable since trnW and trnM-e are expressed in the mitochondria of other land plants. Whereas we could not conclude which tRNA(Met) compensates the lack of expression of trnM-e, we showed that the cytosolic tRNA(Trp) is present in A. thaliana mitochondria, thus compensating the absence of expression of the mitochondrial-encoded trnW.

Characterization of a plant mitochondrial active chromosome.

A method is presented for the partial purification of a plant mitochondrial active chromosome (MAC). This method is based on the presence of the mitochondrial chromosome in the insoluble mitochondrial fraction which allows for its rapid purification from the bulk of detergent-solubilized proteins by ultra-centrifugation. The resuspended MAC carrying DNA and RNA-binding proteins retains DNA synthesis and transcription activities comparable to the ones found in isolated mitochondria. In comparison, tRNA-nucleotidyl terminal transferase taken as an example of RNA modifying activities remains in the soluble fraction. MAC purification is proposed as a rapid and efficient first step in the purification of DNA-binding proteins involved in DNA replication and transcription.

Expression of the two chloroplast-like tRNA(Asn) genes in potato mitochondria: mapping of transcription initiation sites present in the trnN1-trnY-nad2 cluster and upstream of trnN2.

Two copies of the chloroplast-like tRNA(Asn) gene, trnN1 and trnN2, are expressed in potato mitochondria. While Northern-blot analysis revealed only mature tRNA(Asn), RT-PCR indicated that trnN1 is co-transcribed with trnY and nad2 (exons c, d and e). Using primer-extension and capping experiments, four transcription initiation sites have been mapped in the vicinity of these genes. The first site, responsible for the co-transcription of trnN1, trnY and nad2 (exons c, d and e), gives rise to a primary transcript of at least 7000 nt. A second site, 58 nt downstream from trnY, corresponds to an alternative promoter specific for nad2. In both cases, only the CRTA core motif of the consensus CRTAaGaGA of dicot mitochondrial promoters was found. Finally, two transcription initiation sites were identified 135 and 128 nt upstream of trnN2 in a region which shows no sequence homology with this consensus motif.

Differential import of nuclear-encoded tRNAGly isoacceptors into solanum Tuberosum mitochondria.

In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing.

The genes encoding pea and potato mitochondrial tRNAGly and pea mitochondrial tRNASer(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNAGly gene sequences revealed A7:C66 mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNAGly cDNA clones showed that these mispairings are not corrected by C66 to U66 conversions, as observed in plant mitochondrial tRNAPhe. Likewise, a U6:C67 mismatch identified in the acceptor stem of the pea tRNASer(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNACys. In vitro processing reactions with the respective tRNAGly and tRNASer(GCU) precursors show that such conversions are not necessary for 5' and 3' end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.

Structure and expression of several bean (Phaseolus vulgaris) nuclear transfer RNA genes: relevance to the process of tRNA import into plant mitochondria.

Bean nuclear genes for tRNA(Pro), tRNA(Thr) and tRNA(Leu) were isolated. Expression of the tRNA(Pro) genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNA(Pro) gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNA(Thr) genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNA(Thr). The tRNA(Leu) gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNA(Leu) however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNA(Leu) and tRNA(Thr). From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.

Evolutionary aspects of "chloroplast-like" trnN and trnH expression in higher-plant mitochondria.

Two identical "chloroplast-like" tRNAAsn genes, trnN1 and trnN2, have been identified in the potato (Solanum tuberosum) mitochondrial genome. The flanking sequences of trnN1 are unrelated to the corresponding authentic potato chloroplast regions, whilst those of trnN2 are very similar to the chloroplast sequences. The trnN1 copy is present in the mitochondrial genome of various plants whereas the second copy, trnN2, is absent from all the other plant genomes studied so far. Interestingly, both trnN copies are expressed in potato mitochondria. Sequences flanking the chloroplast-like tRNAHis gene (trnH), present as a single copy in the potato mitochondrial DNA, are unrelated to the corresponding chloroplast sequences, whereas chloroplast-derived sequences have been maintained in the vicinity of the maize chloroplast-like mitochondrial trnH gene. However, both the potato and the maize trnH are expressed in mitochondria.

Striking differences in mitochondrial tRNA import between different plant species.

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNA(Phe(GAA)), tRNA(Lys(CUU)), tRNA(Pro(UGG)), tRNA(Ser(GCU)) and tRNA(Ser(UGA))] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNA(His), and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNA(Ser(GCU)) and tRNA(Ser(UGA)). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA import in plants are discussed.

A single editing event is a prerequisite for efficient processing of potato mitochondrial phenylalanine tRNA.

In bean, potato, and Oenothera plants, the C encoded at position 4 (C4) in the mitochondrial tRNA Phe GAA gene is converted into a U in the mature tRNA. This nucleotide change corrects a mismatched C4-A69 base pair which appears when the gene sequence is folded into the cloverleaf structure. C-to-U conversions constitute the most common editing events occurring in plant mitochondrial mRNAs. While most of these conversions introduce changes in the amino acids specified by the mRNA and appear to be essential for the synthesis of functional proteins in plant mitochondria, the putative role of mitochondrial tRNA editing has not yet been defined. Since the edited form of the tRNA has the correct secondary and tertiary structures compared with the nonedited form, the two main processes which might be affected by a nucleotide conversion are aminoacylation and maturation. To test these possibilities, we determined the aminoacylation properties of unedited and edited potato mitochondrial tRNAPhe in vitro transcripts, as well as the processing efficiency of in vitro-synthesized potato mitochondrial tRNAPhe precursors. Reverse transcription-PCR amplification of natural precursors followed by cDNA sequencing was also used to investigate the influence of editing on processing. Our results show that C-to-U conversion at position 4 in the potato mitochondrial tRNA Phe GAA is not required for aminoacylation with phenylalanine but is likely to he essential for efficient processing of this tRNA.

Characterization of the potato mitochondrial transcription unit containing 'native' trnS (GCU), trnF (GAA) and trnP (UGG).

In order to identify the sequences promoting the expression of plant mitochondrial tRNA genes, we have characterized the trnS (GCU), trnF (GAA) and trnP (UGG) transcription unit of the potato mitochondrial genome. These three tRNA genes were shown to be co-transcribed as a 1800 nt long primary transcript. The transcription initiation site located 305 to 312 nt upstream of trnS is surrounded by a purine-rich region but does not contain the consensus motif proposed as a promoter element in dicotyledonous plants. Differential labelling of potato mitochondrial RNA with either guanylyltransferase or T4 polynucleotide kinase suggests that this site corresponds to the unique functional region responsible for the transcription of the three tRNA genes. The initiation site recently found upstream of Oenothera mitochondrial trnF does not seem to be used in potato mitochondria, although a very similar sequence is present 317 nt upstream of the corresponding potato gene. Major processing sites were identified at the 3' end of each tRNA gene. Another processing site, surrounded by a double hairpin structure, is located 498 nt downstream of trnP in stretch of 10 A residues. As judged from northern experiments, this region is close to the determination site of this transcription unit.

Editing and import: strategies for providing plant mitochondria with a complete set of functional transfer RNAs.

The recombinations and mutations that plant mitochondrial DNA has undergone during evolution have led to the inactivation or complete loss of a number of the 'native' transfer RNA genes deriving from the genome of the ancestral endosymbiont. Following sequence divergence in their genes, some native mitochondrial tRNAs are 'rescued' by editing, a post-transcriptional process which changes the RNA primary sequence. According to in vitro studies with the native mitochondrial tRNA(Phe) from potato and tRNA(His) from larch, editing is required for efficient processing. Some of the native tRNA genes which have been inactivated or lost have been replaced by tRNA genes present in plastid DNA sequences acquired by the mitochondrial genome during evolution, which raises the problem of the transcriptional regulation of tRNA genes in plant mitochondria. Finally, tRNAs for which no gene is present in the mitochondrial genome are imported from the cytosol. This process is highly specific for certain tRNAs, and it has been suggested that the cognate aminoacyl-tRNA synthetases may be responsible for this specificity. Indeed, a mutation which blocks recognition of the cytosolic Arabidopsis thaliana tRNA(Ala) by the corresponding alanyl-tRNA synthetase also prevents mitochondrial import of this tRNA in transgenic plants. Conversely, no significant mitochondrial co-import of the normally cytosol-specific tRNA(Asp) was detected in transgenic plants expressing the yeast cytosolic aspartyl-tRNA synthetase fused to a mitochondrial targeting sequence, suggesting that, although necessary, recognition by a cognate aminoacyl-tRNA synthetase might not be sufficient to allow tRNA import into plant mitochondria.

Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs.

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.

Editing corrects mispairing in the acceptor stem of bean and potato mitochondrial phenylalanine transfer RNAs.

Editing is a general event in plant mitochondrial messenger RNAs, but has never been detected in a plant mitochondrial transfer RNA (tRNA). We demonstrate here the occurrence of a tRNA editing event in higher plant mitochondria: in both bean and potato, the C encoded at position 4 in the mitochondrial tRNA(Phe)(GAA) gene is converted into a U in the mature tRNA. This nucleotide change corrects the mismatched C4-A69 base-pair which appears when folding the gene sequence into the cloverleaf structure and it is consistent with the fact that C to U transitions constitute the common editing events affecting plant mitochondrial messenger RNAs. The tRNA(Phe)(GAA) gene is located upstream of the single copy tRNA(Pro)(UGG) gene in both the potato and the bean mitochondrial DNAs. The sequences of potato and bean tRNA(Pro)(UGG) genes are colinear with the sequence of the mature bean mitochondrial tRNA(Pro)(UGG), demonstrating that this tRNA is not edited. A single copy tRNA(Ser)(GCU) gene was found upstream of the tRNA(Phe) gene in the potato mitochondrial DNA. A U6-U67 mismatched base-pair appears in the cloverleaf folding of this gene and is maintained in the mature potato mitochondrial tRNA(Ser)(GCU), which argues in favour of the hypothesis that the editing system of plant mitochondria can only perform C to U or occasionally U to C changes.

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species.

The initiator methionine transfer RNA (tRNA(fMet)) gene was identified on a 347 bp Eco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAs(fMet) or tRNA(fMet) genes studied so far. Whereas the tRNA(fMet) gene is present as a single copy in the potato mt genome, a tRNA 'pseudogene' corresponding to 60% of a complete tRNA (from the 5' end to the variable region) and located at 105 nucleotides upstream of the tRNA(fMet) gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNA(fMet) gene in the mt genome is very different among plants, which suggests that the tRNA(fMet) gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Evidence that nuclear-encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.