Search

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 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.

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11-12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA(Phe) species, namely tRNA(Phe)1 and tRNA(Phe)2. By sequence determination, we show that tRNA(Phe)2 is identical to the previously sequenced tRNA(Phe)1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs(Phe) vary during leaf development: in etiolated leaves the undermodified tRNA(Phe)2 only represents 15% of total chloroplast tRNA(Phe), during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA(Ala)(U*GC) cp tRNA(Pro)(U*GG) and cp tRNA(Arg)(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that 'U:U and U:C wobble' mechanisms can operate to allow the reading of these codons by cp tRNA(Leu)(UAm7G).

The genes coding for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) have been identified in Oenothera mitochondrial DNA. Sequence analysis of these genes and their surrounding sequences are presented and compared with other known tRNA genes from plant mitochondria. All six deduced tRNA sequences can be folded into the classical cloverleaf structure model. Only the tRNA(His) gene shows high homology with the corresponding chloroplast gene and thus appears to be derived from a transfer event of chloroplast sequences into the mitochondrial genome. The sequences surrounding this gene, however, show little similarity with the chloroplast genome. The other five deduced tRNAs display a much lower similarity with their chloroplast counterparts and thus appear to be genuine mitochondrial tRNAs. These tRNAs are highly conserved between monocots and dicots with maximally three nucleotides differing between the Oenothera sequences and their wheat homologues. A purine-rich sequence is found upstream of each tRNA gene in Oenothera, similar to wheat mitochondrial tRNA genes, that could be involved in transcription signalling.

The nucleotide sequences of soybean chloroplast tRNAsLeu were determined using post-labeling techniques. Comparison of the primary structures of soybean chloroplast tRNAsLeu with their bean, maize and spinach counterparts only show few base differences. Contrary to previously published results (1) a re-examination of bean tRNALeu sequence shows that this tRNA resembles soybean and maize tRNA2Leu in structure.

The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme). The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme. There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme. Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. There are two tRNAsLys in the cytoplasm. Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E. coli enzyme. Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E. coli enzyme. Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E. coli enzyme.

A cluster of three tRNA genes located on fragment Bam6a from Vicia faba chloroplast DNA has been sequenced: it contains the genes for tRNA(CAALeu), tRNA(UAALeu) and tRNA(Phe). The two tRNA(Leu) genes are separated by 443 bp and are transcribed divergently from different DNA strands. The intergenic region contains a series of short repeats and a partial copy of the split tRNA(UAALeu) gene which includes 100 bp of the 5' flanking region, 35 bp of the 5'exon and the first 42 bp of the intron. It is possible that some of these duplications occurred upon the rearrangement of the two tRNA(Leu) genes in broad bean (and in pea) or upon the deletion of one copy of the inverted repeat, since in all other higher plant chloroplast genomes studied so far these two tRNA(Leu) genes are located far apart on the genome, one being in the inverted repeat region, the other one in the large single copy region. The tRNA(Phe) and tRNA(UAALeu) are encoded by the same DNA strand, and separated by 110 bp.

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.

Bean mitochondria and chloroplast tRNAsTrp, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced using post-labeling techniques. The high degree of sequence homology between bean mitochondria and chloroplast tRNAsTrp shows that these two tRNAs are coded for by closely related genes which have probably evolved from a common ancestor gene. The anticodon of bean mitochondria tRNATrp is CmCA, which can recognize UGG (the codon for tryptophan in the universal code) and is complementary neither to UGA (which codes for tryptophan in mammalian and yeast mitochondria) nor to CGG (which could be a tryptophan codeword in plant mitochondria).

We report the nucleotide sequence of three tRNA genes from maize mitochondria. The genes are located in two BamHI fragments, 3.55 and 5.7 kb long, adjacent to the S2 sequence in the maize mitochondrial genome. On the 3.55 kb BamHI fragment, we have characterized a tRNA(Cys)(GCA) gene. A strong sequence homology of this tRNA(Cys)(GCA) gene with its chloroplast counterpart in wheat suggests that it may be part of a chloroplast DNA insertion into the mitochondrial genome. This gene has been found to be transcribed in the mitochondrion. Two tRNA genes are located on the 5.7 kb BamHI fragment, separated from each other by 250 bp. One is a mitochondrial tRNA(Ser)(GCU) gene. The other, a non-transcribed tRNA(Phe)-like gene, is interrupted by a 49 base-pair inserted DNA sequence in the variable loop and has a Leu (UAA) anticodon.

Three tRNA genes have been isolated from a genomic library of Arabidopsis thaliana: a tRNASer (GCU), a tRNATyr (GUA) and a tRNAGlu (UUC) genes. These genes are located closely on the same DNA fragment. The tRNASer and the tRNAGlu genes have both 99% sequence similarity with their mitochondrial counterparts from higher plants indicating that these three tRNA genes are mitochondrial. The tRNATyr gene shows a particular high sequence similarity with the mitochondrial tRNAPhe pseudogene from maize, and both genes are flanked by a tRNASer gene in the upstream region. Extensive sequence comparisons of the Arabidopsis thaliana mitochondrial sequence containing the three tRNA genes and the corresponding region from maize and soybean mitochondria have shown evidence that the tRNA Tyr gene has been generated from a mitochondrial tRNAPhe gene. The conversion was accomplished by three genetic events: a 4 base-pair deletion, a mutation and a recombination, which led to the transformation of the acceptor stem and the anticodon.

The entire nucleotide sequence of a 2014 bp BamH I fragment from broad bean (Vicia faba) chloroplast DNA containing the genes for tRNAThr (trnT), tRNAGlu (trnE) and tRNATyr (trnY) has been determined. The tRNAGlu and tRNATyr genes are separated by only 60 bp and are probably part of the same transcriptional unit. The tRNAThr gene is located on the complementary strand, 876 bp away from the tRNAGlu gene. This fragment also contains an open reading frame of 82 codons, as well as a series of AT-rich, direct and inverted repeats.

We have used bean mitochondrial (mt) and chloroplast (cp) tRNA(Trp) as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNA(Trp) genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA(Trp) genes are 97% homologous. On the wheat cp DNA, a tRNA(UGGPro) gene was found 139 bp upstream of the cp tRNA(Trp) gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA(Pro) gene was found 136 bp upstream of the mt tRNA(Trp) gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA(Trp) gene separated by about 140 bp from a tRNA(Pro) gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA(Trp) gene and the tRNA(Pro) (or partial tRNA(Pro)) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence. Wheat mt tRNA(Trp) gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria.

Leucyl-tRNA synthetase from Phaseolus vulgaris chloroplasts could be separated from its cytoplasmic counterpart upon chromatography on hydroxyapatite, but the cytoplasmic and mitochondrial leucyl-tRNA synthetases could not be distinguished. The tRNALeu species from the various plant cell compartments and from Escherichia coli were aminoacylated using either homologous or heterologous enzymes; the levels of aminoacylation and the profiles of the leucyl-tRNAs upon reverse-phase chromatography were studied. Cytoplasmic tRNALeu species could be aminoacylated by the cytoplasmic or by the mitochondrial enzymes and in both cases yielded two peaks upon reverse-phase chromatography (RPC-5). But they could not be charged by the chloroplast-specific or by the E. coli enzynes. Mitochondrial tRNALeu species could be charged by the mitochondrial or by the cytoplasmic enzymes and in both cases yielded four peaks upon reverse phase (RPC-5) chromatography. But they could not be aminoacylated using the chloroplast-specific or the E. coli leucyl-tRNA synthetases. Chloroplastic tRNALeu species can be divided into two classes: the first class contains four isoacceptor species which can be charged by the cytoplasmic or mitochondrial enzymes, but not by the chloroplast-specific or the E. coli enzymes; the second class contains three chloroplast-specific tRNALeu species which can be charged by the chloroplast-specific or the E. coli enzymes but not by the cytoplasmic or the mitochondrial enzymes. There are five isoacceptor tRNALeu species in E. coli; all are charged by the E. coli or the chloroplast-specific enzymes, while only one is aminoacylated by the plant cytoplasmic or mitochondrial enzymes.

Met-tRNA synthetase from Paseolus vulgaris cytoplasm could be separated from its chloroplastic or mitochondrial counterpart by DEAE-cellulose chromatography, but the Met-tRNA synthetase from the two latter organelles could not be distinguished using DEAE-cellulose, hydroxyapatite or CM-Sephadex chromatography. As revealed by reverse-phase chromatography, bean cytoplasm contains 2 tRNAsMet; only one is charged by chloroplast, mitochondrial or Escherichia coli Met-tRNA synthetase. Mitochondria contain, in addition to the 2 cytoplasmic tRNAsMet, 3 mitochondria-spedific tRNAsMet; 2 can be formylated by the mitochondrial or the E. coli transformylase; all 3 are charged by mitochondrial, chloroplastic or E, coli Met-tRNA synthetase; none is charged by the cytoplasmic enzyme. Chloroplasts contain, in addition to the 2 cytoplasmic tRNAsMet, 3 chloroplast-specific tRNAsMet, different from the mitochondrial tRNAsMet; one is formylatable by the chloroplastic or the E. coli transformylase; all 3 are charged by chloroplastic, mitochondrial or E. coli Met-tRNA synthetase; only one is charged by the cytoplasmic enzyme. Of the 3 E. coli tRNAsMet, only the formylatable species can be charged by bean cytoplasmic, chloroplastic or mitochondrial Met-tRNA synthetase.

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules. Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.

The sequence of maize chloroplast tRNAIle2, encoded in the 16S to 23S rDNA spacer, was determined using in vitro labeling techniques. The sequence is: pG-G-G-C-U-A-U-U-A-G-C-U-C-A-G-U-Gm-G-D-A-G-A-G-C-m22G-C-G-C-C-C-C-U-G-A-U-t6A- A-G-G-G-C-G-A-G-m7G-acp3U-C-U-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-G-A-U-G-G-C-C-C-A -C-C-AOH. This sequence is identical to that predicted from the corresponding gene sequence, after excision of a long intervening sequence (1), but shows the post-transcriptional modifications of this tRNA. Furthermore it demonstrates that the excision of the intron occurs after the second base following the anticodon and that this gene, which is over 1000 base-pair long, is transcribed and processed into a mature functional chloroplast-tRNA. The sequence of maize (a monocot) and spinach (a dicot) tRNAIle2 are shown to be identical.

The review sums up data on gene mapping studies of tRNAs of chloroplasts from maize, beans, Euglena, Cyanophora. The mechanisms of splicing of tRNA2Ile from maize chloroplasts and coded for by a gene of unusual length was investigated.

A region of about 2 kb which is almost identical in the wheat and maize mitochondrial genomes has been sequenced. It contains a tRNA(Ser) gene, a pseudo-tRNA gene and two open reading frames coding for subunit 3 of the NADH dehydrogenase (118 amino acids) and for ribosomal protein S12 (125 amino acids). The two protein genes are separated by 47 bp and are co-transcribed in wheat and maize. Two transcripts of about 0.9 kb and 3.0 kb, each coding for both proteins, have been characterized, but no monocistronic transcript was detected. Each gene is preceded by a putative ribosome binding site. The pseudo-tRNA gene is interrupted by two insertion sequences in wheat and by one in maize. The origin of the additional interrupting sequence found in the wheat pseudo-tRNA gene, which is also present elsewhere in the mitochondrial genomes, is discussed.

The genes for the initiator tRNA(Met)CAU, tRNA(Gly)UCC, tRNA(Thr)GGU, tRNA(Glu)UUC and tRNA(Tyr)GUA and an open reading frame of 62 codons have been identified by sequencing a 2,358 bp BamHI and a 1,378 bp BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 bp intergenic region between the genes for the initiator tRNA(Met) and the gene for tRNA(Gly)UCC is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 bp of the intergenic region and the first 28 bp of the tRNA(Met) gene, the second copy is found downstream of the tRNA(Met) gene.