Wheat Mitochondria Research Articles

Identification of a nuclear-encoded sunflower mitochondrial tRNA-Leu (AAG)

Transfer RNA molecules present in higher-plant mitochondria have three distinct genetic origins. Some are coded by mitochondrial genes having 65-80~o similarity with the corresponding eubacterial and chloroplast genes. They are classified as 'native' tRNA genes [4]. Other tRNA molecules are coded by mitochondrial genes which show a sequence similarity of 90 -100~ with the homologous chloroplast genes. These genes are classified as chloroplast-like tRNA genes and are believed to be part of chloroplast DNA insertions in the mitochondrial genomes [4]. A third group of mitochondrial tRNA molecules are encoded in the nuclear genome and imported into the mitochondria, as has been demonstrated in bean for eight mitochondrial tRNAs [7]. Four of the bean tRNAs have been identified as tRNA-Leu and three of them have also been sequenced [2, 5, 7]. The same situation also seems to occur in wheat and potato mitochondria where, using different approaches, several tRNAs highly homologous to their cytoplasmic counterparts have been detected [4, 6]. In order to verify the existence of nuclearencoded mitochondrial tRNAs in other higher plants, we started their identification in sunflower. Highly purified total tRNAs from sunflower mitochondria were isolated by the method of Marechal-Drouard et al. [7]. A specific oligonucleotide (5 ' T G T C A G A A G T G G G A T T T G A A CCCA-3'), complementary to the last 24 nucleotides of the bean mitochondrial tRNA-Leu (NAA) [2], was used as primer in a reverse transcription assay carried out using 7.5 U of AMV reverse transcriptase (Promega Biotec.), in the presence of 2 #g of sunflower mitochondrial tRNAs as a template. Incubation conditions were as suggested by the supplier. The reverse transcriptase-generated product was amplified by Taq polymerase enzyme (Promega Biotec.) in the presence of two 24-mer oligonucleotides (5 ' G T C A G G A T G G C C G A G TGGTCTAAG-3 ' and 5 ' -TGTCAGAAGTGGGATTTGAACCCA-3 ' ), corresponding to the 5' and 3' termini of the bean mitochondrial tRNALeu (NAA) [ 2] respectively. The products of both the reverse transcription and PCR amplification reactions are shown in Fig. 1.

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.

Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites

To identify transcription initiation sites in wheat mitochondria, the nascent 5'-ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [alpha-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed.

Isolation from wheat mitochondria of a membrane-associated high molecular weight complex involved in DNA synthesis

A high molecular weight mitochondrial DNA (mtDNA) replication complex, associated with the mitochondrial membrane, was isolated by sucrose gradient centrifugation from purified wheat embryo mitochondria. This complex comprised the mtDNA as well as enzyme activities involved in the replication and transcription of the organelle genome, such as DNA polymerase, RNA polymerase and topoisomerase type I. The isolated complex is active in mtDNA and mtRNA synthesis in vitro. Electron microscopy and lipid analysis confirmed the membrane origin of this complex. Enzyme activities are resistant to physiological ionic strengths, 0.1-0.2 M KC1, while the membrane-mtDNA association is resistant up to 1 M KC1. DNase treatment of the complex released the DNA polymerase activity while protease treatment solubilized mtDNA, suggesting the direct interaction of mtDNA with membrane protein(s). The use of a novel approach to detect mtDNA fragments specifically retained by the mitochondrial membranes after Sal I digestion of the complex suggests that specific mtDNA sequences anchor mtDNA to mitochondrial membranes.

RNA editing of the transcript coding for subunit 4 of NADH dehydrogenase in wheat mitochondria: uneven distribution of the editing sites among the four exons

The wheat mitochondrial (mt) NADH dehydrogenase subunit 4 gene (nad4) has been localized and sequenced. This gene, about 8 kb long, is composed of four exons separated by three class II introns. The nad4 gene exists as a single copy in the wheat mitochondrial genome and it is transcribed into one abundant mRNA of 1.8 kb, whose extremities have been mapped. The complete cDNA sequence corresponding to the nad4 transcript has been determined by combining the direct sequencing of uncloned cDNA and a method involving cDNA synthesis and PCR amplification using specific oligonucleotides as primers, followed by cloning and sequencing of the amplification product. Comparison of the genomic sequence with that of the cDNA shows that all nad4 transcripts are fully edited at 23 positions, with an uneven distribution of the editing sites between the different exons: While exon 1 and exon 4 are extensively edited (with a change of 11% of the amino acid sequence), exon 2 is not edited at all and exon 3 is 0.5% edited. This uneven distribution is discussed.

In vitro processing of transcripts containing novel tRNA-like sequences (?t-elements?) encoded by wheat mitochondrial DNA

We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.

Structure and transcription of the gene coding for subunit 3 of cytochrome oxidase in wheat mitochondria

The wheat mitochondrial (mt) cox3 has been localized and sequenced. The gene exists as a single copy in the wheat mt master chromosome and is transcribed into a single 1.2 kb RNA, whose extremities have been mapped. Comparison of the wheat and Oenothera cox3 sequences gives ambiguous indications concerning the amino acid coded by the codon CGG. Upstream and downstream of the wheat cox3 gene, two short sequences of 43 bp and 69 bp respectively are present, which are almost identical to sequences present in the flanking regions of other plant mitochondrial genes. These common sequences seem to have played a role in the rearrangements which caused sequence divergence of the plant mt genomes during evolution. Furthermore, mapping of wheat and maize cox3 and cob transcripts suggests that some of these common sequences can play a role in the regulation of transcription or processing.

RNA editing at a splicing site of NADH dehydrogenase subunit IV gene transcript in wheat mitochondria

Comparison between the sequence of the gene coding for the wheat mitochondrial NADH dehydrogenase subunit IV (nad4) and the cDNA sequence obtained by reverse transcription, using total wheat mtRNA as template, has shown the presence of a uridine residue, not encoded by the genomic sequence, at the exon2-exon3 junction of the spliced transcript. This U creates a non-encoded CUG leucine codon which is essential for maintaining the reading frame, as shown by the conservation of the amino acid sequence of the NAD4 protein in various species. The addition of a U or the specific post-transcriptional conversion of a C to a U could explain this phenomenon.

RNA editing in plant mitochondria

A basic principle of molecular biology is that the primary sequence of RNA faithfully reflects the primary sequence of the DNA from which it is transcribed. This concept has been challenged recently by the discovery of RNA editing, broadly defined as any process that changes the nucleotide sequence of an RNA molecule from that of the DNA template encoding it. Examples of RNA editing (see ref. 2 for review) include the insertion and deletion of uridine residues in mitochondrial messenger RNAs in kinetoplastid protozoa, the conversion of a cytidine to uridine in mammalian apolipoprotein-B mRNA, and the appearance of two non-templated guanosine residues in a paramyxovirus transcript. In these cases, RNA editing either re-tailors a non-functional transcript, producing a translatable mRNA, or modifies an already functional mRNA so that it generates a protein of altered amino-acid sequence. Here we report an editing phenomenon that involves the conversion of cytidine to uridine at multiple positions in the mRNA for subunit II of cytochrome c oxidase in wheat mitochondria. Such RNA editing provides an explanation for apparent coding anomalies in plant mitochondria.

Aspartate and asparagine tRNA genes in wheat mitochondrial DNA: a cautionary note on the isolation of tRNA genes from plants.

We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpińska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulińska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.

Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis.

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants.

The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene. The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene.

The mitochondrial S13 ribosomal protein gene is silent in wheat embryos and seedlings

The sequence of a wheat mitochondrial reading frame encoding a protein homologous to the E. coli S13 small subunit ribosomal protein has been determined. The gene is located immediately downstream of a 1.4 kb recombinationally-active repeat element that contains the ATPase subunit 6 gene. The coding regions of the two genes are separated by only 153 bp, the shortest distance yet observed between protein-coding genes in plant mitochondria. However, their transcript profiles differ markedly. The ATPase 6 gene displays a single, prominent mRNA of approximately 1.4 kb, whereas the S13 gene shows no stable transcript as judged by Northern blot analysis of wheat mitochondrial RNA isolated from different developmental stages. A short segment of the 26S rRNA gene is located downstream of the S13 gene and its presence illustrates the frequent DNA duplication/rearrangements found in wheat mitochondria.

A DNA topoisomerase type I from wheat embryo mitochondria

In order to study DNA replication and expression in wheat mitochondria our laboratory has been seeking to develop a system that supports DNA synthesis and transcription, either in isolated mitochondria from wheat embryos or in a mitochondrial lysate from the same source deprived of endogenous DNA in vitro. We have characterized some of the enzymes involved in the DNA synthesis and transcription process. In this study we describe a DNA topoisomerase activity.Broken mitochondria from wheat embryos can actively relax negatively supercoiled DNA (pBR322, pAT153, etc...). The enzyme is intramitochondrial: the activity is detected only when intact organelles are broken by non-ionic detergent. Most of the topoisomerase activity found in the broken mitochondria is recovered in the mitochondrial lysate. It is stimulated by Mg(2+) and has an optimum salt concentration, KCl or NaCl, between 50 mM and 100 mM. ATP has no effect on this activity. Ethidium bromide, berenil, novobiocine and nalidixic acid, compounds currently used to characterize DNA topoisomerases, do not effect the relaxation of supercoiled DNA by the wheat mitochondrial activity. On the other hand N-ethylmaleimide has a strong inhibitory effect indicating that sulfhydryl groups are essential for enzyme activity. The molecular weight of the enzyme as determined by glycerol gradient sedimentation, is about 110 kd. Another important feature of the mitochondrial lysate DNA topoisomerase is the ability to relax positively supercoiled DNA, a property of eukaryotic topoisomerases I.

Wheat mitochondrial 26S ribosomal RNA gene has no intron and is present in multiple copies arising by recombination

Analysis of cloned SalI fragments coding for the 26S rRNA in wheat mitochondria (Triticumaestivum L.) shows that the 26S gene is contained within the same basic structural unit RS (which is about 6.6 kbp long). This repeated sequence is flanked by four different sequences I, II, III, IV in the orientations: I-RS-III, I-RS-IV, II-RS-III and II-RS-IV. This organization of the 26S gene, similar to the organization found for the 5S and 18S gene, suggests again that reciprocal intra-and/or intermolecular recombination occurs within these repeated sequences. S1 mapping and R loop mapping indicate that the 26S gene is about 3.5 kbp and has no intron.

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non-conservative ones. The wheat COII gene has a highly-conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron-insert sequences. We find that a sequence identical to the region of the 5' exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity.

Small circular DNA molecules in wheat mitochondria

The presence of polydisperse small circular DNAs in wheat cells was first confirmed by the mica-pressadsorption (MPA) method for electron microscopy. To identify their location in the cell, chloroplast and mitochondrial fractions were examined separately by the same method; small circular DNAs were scarcely found in the former but abundantly in the latter fraction, indicating their origin from mitochondria. The size varied greatly, ranging from 0.1 to 2.0 μm in contour length. To verify the present finding, the same mitochondrial fraction was examined by the conventional cytochrome-spreading method by which the presence of the same size-class of circular DNAs was confirmed.

Unique species of 5 S, 18 S, and 26 S ribosomal RNA in wheat mitochondria

Unique species of 5 S, 18 S, and 26 S ribosomal RNA in wheat mitochondria

Studies of the functional activity of mitochondria and cell ultrastructure of the coleoptile in wheat, Agropyron and their hybrids (incomplete amphidiploids) during decreasing temperature

The structure of the mitochondria and endoplasmic reticulum in the coleoptile of plants (Triticum aestivum var. Lutescens 329, Agropyron glaucum, Triticum × Agropyron 56-chromosome hybrids, incomplete amphidiploids, TAH, containing 42 wheat chromosomes and 14 chromosomes of genomes D or X of Agropyron), which differ in winterhardiness, was studied after exposure to 0 ° and -4 °C for periods varying from 10 min to 4 days. The functional activity of mitochondria isolated from 3 day old seedlings was also investigated in these cereals. The cells of Triticum and Agropyron seedlings grown at 23 °C were shown to differ in mitochondrial structure. In the cells of TAH both kinds of mitochondria were found.On day 4 of exposure to -4 °C, the mitochondria of Agropyron cells were not changed; the endoplasmic reticulum formed complex closed cavities. Under similar conditions most wheat mitochondria were destroyed and in the rest no cristae were observed.Morphometric analysis indicated that the volume of such mitochondria increases by two times, while the surface area of the internal membranes and cristae decreases by 1.54 times. In such cells, the endoplasmic reticulum is represented only by membranes of the smooth type. The structure of the mitochondria and endoplasmic reticulum in the seedling cells of TAH 829, which is more like Agropyron in winterhardiness, is similar to that of Agropyron cells; in hybrid 822 (more like wheat containing the D genome), changes arise resembling those observed in wheat. The existence of different types of mitochondria in seedling cells of TAH is especially distinct at low temperatures.The mitochondria of the cereals studied differ in biochemical activity after low temperature treatment (0 ° and - 4 °C). Phosphorylative and oxidative activity of mitochondria of the winterhardy forms (Agropyron glaucum, TAH 829) decreases just after the beginning of low temperature treatment. At the same time, the morphology of the mitochondria undergoes reversible changes. The mitochondria of cold-susceptible forms of wintering plants (Triticum aestivum, TAH 822) do not conform to this pattern. Under long-term low temperature treatment they display irreversibly damaged mitochondria. It is suggested that the winterhardy forms have high adaptability connected with a rapid protective response of the cell mitochondria and endoplasmic reticulum. This adaptability is regulated by nuclear genes: TAH have different mitochondria in the coleoptile cells; if genome X of Agropyron is present, which TAH derives from the male parent, the related mitochondria become more resistant to low temperature treatment.

Pathways for the oxidation of malate and reduced pyridine nucleotide by wheat mitochondria.

Piericidin A and oxaloacetate have been used as inhibitors to investigate the operation and localisation of NAD+‐linked dehydrogenases and the possible compartmentation of these enzymes within the matrix of the mitochondrion. Mitochondria isolated from etiolated shoots of wheat (Triticum vulgare) were shown to oxidise a variety of NAD+‐linked substrates. Oxaloacetate, when added to rapidly respiring preparations (i.e. state 3) resulted in a large but transient inhibition of the oxidation of both pyruvate and citrate, but had little effect on the rate of oxidation of malate. The products of oxidation of malate, in the absence of added NAD+, were found to be a large quantity of pyruvate together with smaller quantities of oxaloacetate and citrate. Using potassium ferricyanide as a non‐penetrating electron acceptor it was possible to show that the malate oxidation occurred in the matrix. This is consistent with the operation of both the malic enzyme and a low level of malate dehydrogenase within the matrix of the mitochondrion. The low level of inhibition of malate oxidation by oxaloacetate was a function of the concentration of malate and appeared to be of a competitive nature. Measurement of the redox state of the endogenous NAD+ pool showed that under phosphorylatig conditions both malate and citrate reduce approximately 15% of the total coenzyme. The addition of 0.2 mM oxaloacetate to phosphorylating aerobic mitochondria failed to oxidise any of the NAD+ reduced by malate but resulted in a partial oxidation of NAD+ reduced by citrate. The transition from the aerobic to anaerobic state in the presence of malate resulted in a biphasic reduction of the endogenous NAD+, whereas in the presence of citrate it was monophasic. The oxidation of NAD+‐linked substrates, including malate, was found to be only partially inhibited by piercidin A. The piericidin‐A‐resistant pathway was found to be located internally on the basis of experiments using ferricyanide as a non‐penetrating electron acceptor. The first site of phosphorylation was not coupled to oxidation mediated by the piericidin‐A‐resistant pathway. The presence of piericidin A altered the shape of the malate saturation curve. In the absence of the inhibitor it was found to be biphasic; saturating at 60 mM and in its presence to be monophasic saturing at 15 mM. At high concentrations of malate (60 mM) piericidin A caused approximately a 30% inhibition, similar to that found with other NAD+‐linked substrates, while at lower concentrations of malate (15 mM) piericidin A caused a much higher inhibition (80%), which subsequently recovered to the much higher piericidin‐A‐resistant rate observed with higher concentrations of malate. In the presence of piericidin A oxaloacetate caused severe but transient inhibition of malate oxidation, similar in magnitude to that observed during pyruvate oxidation in the absence of piericidin A. It was concluded that the data were consistent with the spatial separation of both the internal malic enzyme and malate dehydrogenase together with the internally located piericidin‐A‐sensitive and resistent electron‐transport pathways.