The HIV-1 Pr55 gag polyprotein binds to plastidial membranes and leads to severe impairment of chloroplast biogenesis and seedling lethality in transplastomic tobacco plants.

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

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.

Role of editing in plant mitochondrial transfer RNAs.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base pair in the acceptor stem of mt tRNA(Phe) (GAA). In larch mitochondria, three C to U editing events restore U:A base pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNA(His) (GUG). For both these mt RNA(Phe) and tRNA(His), editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNA(Cys) (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base pair, and reverse transcriptase minisequencing has shown that the mature mt tRNA(Cys) is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of a U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor, nor aminoacylation of tRNA(Cys), requires C to U editing at this position. But sequencing of the purified mt tRNA(Cys) has shown that Psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into Psi at position 28.

Editing of plant mitochondrial transfer RNAs.

Editing in plant mitochondria consists in C to U changes and mainly affects messenger RNAs, thus providing the correct genetic information for the biosynthesis of mitochondrial (mt) proteins. But editing can also affect some of the plant mt tRNAs encoded by the mt genome. In dicots, a C to U editing event corrects a C:A mismatch into a U:A base-pair in the acceptor stem of mt tRNAPhe (GAA). In larch mitochondria, three C to U editing events restore U:A base-pairs in the acceptor stem, D stem and anticodon stem, respectively, of mt tRNAHis (GUG). For both these mt tRNAs editing of the precursors is a prerequisite for their processing into mature tRNAs. In potato mt tRNACys (GCA), editing converts a C28:U42 mismatch in the anticodon stem into a U28:U42 non-canonical base-pair, and reverse transcriptase minisequencing has shown that the mature mt tRNACys is fully edited. In the bryophyte Marchantia polymorpha this U residue is encoded in the mt genome and evolutionary studies suggest that restoration of the U28 residue is necessary when it is not encoded in the gene. However, in vitro studies have shown that neither processing of the precursor nor aminoacylation of tRNACys requires C to U editing at this position. But sequencing of the purified mt tRNACys has shown that psi is present at position 28, indicating that C to U editing is a prerequisite for the subsequent isomerization of U into psi at position 28.

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.

Mitochondrial tRNA import: are there distinct mechanisms?

Sequence information from an increasing number of complete mitochondrial genomes indicates that a large number of evolutionary distinct organisms import nucleus-encoded tRNAs. In the past five years, much research has been initiated on the features of imported tRNAs, the mechanism and the energetics of the process as well as on the components of the import machinery. In summary, these studies show that the import systems of different species exhibit some unique features, suggesting that more than one mechanism might exist to import tRNAs.

Expression of Arabidopsis thaliana mitochondrial alanyl-tRNA synthetase is not sufficient to trigger mitochondrial import of tRNAAla in yeast.

It has often been suggested that precursors to mitochondrial aminoacyl-tRNA synthetases are likely carriers for mitochondrial import of tRNAs in those organisms where this process occurs. In plants, it has been shown that mutation of U(70) to C(70) in Arabidopsis thaliana tRNA(Ala)(UGC) blocks aminoacylation and also prevents import of the tRNA into mitochondria. This suggests that interaction of tRNA(Ala) with alanyl-tRNA synthetase (AlaRS) is necessary for import to occur. To test whether this interaction is sufficient to drive import, we co-expressed A. thaliana tRNA(Ala)(UGC) and the precursor to the A. thaliana mitochondrial AlaRS in Saccharomyces cerevisiae. The A. thaliana enzyme and its cognate tRNA were correctly expressed in yeast in vivo. However, although the plant AlaRS was efficiently imported into mitochondria in the transformed strains, we found no evidence for import of the A. thaliana tRNA(Ala) nor of the endogenous cytosolic tRNA(Ala) isoacceptors. We conclude that at least one other factor besides the mitochondrial AlaRS precursor must be involved in mitochondrial import of tRNA(Ala) in plants.

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.

Compilation and analysis of plant mitochondrial promoter sequences: An illustration of a divergent evolution between monocot and dicot mitochondria.

We have analyzed 67 sequences surrounding transcription initiation sites identified in higher plant mitochondria. The sequences were classified, independently for monocots and dicots, according to the presence of the CRTA core element found upstream of the first transcribed nucleotide and previously reported as an essential element of plant mitochondrial consensus promoters. This compilation provides new elements concerning the structure of consensus promoters and the relative importance of non-conserved promoters in plant mitochondria. It can be emphasized that promoter regions exhibit several differences between monocot and dicot mitochondria, presumably reflecting a divergent evolution: The sequences classified among consensus promoters as well as the distance between the first transcribed nucleotide and the core element are highly conserved in dicots while more plasticity is observed in monocots. It also appears that the proportion of promoters with neither the conserved promoter sequence nor any conserved motif is far greater in dicots than in monocots.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana.

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl-tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl-tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl-tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

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.

A promoter element active in run-off transcription controls the expression of two cistrons of nad and rps genes in Nicotiana sylvestris mitochondria.

The expression of two mitochondrial gene clusters (orf87-nad3-nad1/A and orf87-nad3-rps12) was studied in Nicotiana sylvestris. 5' and 3' termini of transcripts were mapped by primer extension and nuclease S1 protection. Processing and transcription initiation sites were differentiated by in vitro phosphorylation and capping experiments. A transcription initiation site, present in both gene clusters, was found 213 nucleotides upstream of orf87. This promoter element matches the consensus motif for dicotyledonous mitochondrial promoters and initiates run-off transcription in a pea mitochondrial purified protein fraction. Processing sites were identified 5' of nad3, nad1/A and rps12 respectively. These results suggest that (i) the expression of the two cistrons is only controlled by one duplicated promoter element, and (ii) multiple processing events are required to produce monocistronic nad3, nad1/A and rps12 transcripts.