Topic 11.6

The Genetic System in Plant Mitochondria Has Some Special Features

Allan G. Rasmusson, Lund University, Sweden; Ian M. Møller, Royal Veterinary and Agricultural University, Denmark

As in plastids, the genetic system in mitochondria differs both structurally and functionally from the nuclear genome and its gene expression. On the other hand, mitochondria share several genetic features with bacteria. For example, several genes are transcribed together and the ribosomes are of a prokaryotic type. This is in line with the evolutionary origin of the mitochondrion as an endosymbiont of the α-subclass of Proteobacteria, a group including the intracellular pathogen Ricketsia prowazekii (Gray et al. 1999).

Plants are also distinctly different from other eukaryotes in their mitochondrial DNA (mtDNA) and its expression. For example, the plant mtDNA is very large and highly variable in size between species (Backert et al. 1997). The presence of several repeats enables the genome to recombine, changing the gene order or even causing a split into smaller subgenomic molecules, each carrying a subset of the genes (Figure 1). Though the size of mtDNA varies between plants, the number of genes appears to be similar in all plants. In the fully sequenced 367 kilobasepair (kb) mtDNA of Arabidopsis thaliana (Unseld et al. 1997; Marienfeld et al. 1999), the known genes (constituting 65 kb) are scattered in a background of noncoding sequence. A similar situation can be found in the sequenced mtDNA of sugarbeet (Kubo et al. 2000). Plant mtDNA is quite different from the much smaller (16 kb) single mtDNA molecule of mammals in which every base pair has a function in coding for protein or mediating transcription and replication, and neither introns nor repeats exist.

Figure 1 Recombination in mtDNA of Arabidopsis thaliana. The mtDNA contains a direct repeat (I) and an inverse repeat (II). Recombination at the two repeats will change the gene order and also split up the genome into two separate molecules. (After Unseld et al. 1997.) (Click image to enlarge.)

The expression of the mitochondrial genes in plants is carried out by RNA polymerases that are unlike the nuclear polymerases, but related to the enzymes in bacteriophages. For several mitochondrial genes, these phage-type polymerases start up transcription from multiple promoters (Kühn et al. 2005). Many genes in plant mtDNA are broken up into exons by introns. The introns are removed in the processing of the primary RNA into a mature mRNA that can be translated into the correct polypeptide (Figure 2). The introns in mitochondria belong to the so-called self-splicing type II group, in which splicing is defined by the intron structure. The splicing can also work in the trans mode, in which it can join segments from separate RNA molecules, and thus assemble protein-coding reading frames even if the exons are distributed on different primary transcripts. This allows individual exons of a gene to reside independently in the genome, even in different subgenomic DNA molecules. Because mitochondrial genes are often co-transcribed with unrelated genes into a polycistronic primary transcript, RNA processing also includes endonucleolytic reactions, so that each mature mRNA encodes only one protein (see Figure 2). Like mRNAs for nuclear encoded genes, mitochondrial mRNAs are polyadenylated. However, instead of stabilizing (the case for nuclear encoded mRNAs), the mitochondrial mRNA polyadenylation mediates degradation. Instead, stem-loops stabilise the mRNA in plant mitochondria (Gagliardi and Leaver 1999; Kuhn et al. 2001).

Figure 2 Mitochondrial gene expression. The mitochondrial nad2 gene in Arabidopsis thaliana encodes the NAD2 subunit of respiratory complex I. The figure shows the RNA processing steps needed for this gene to be expressed into an mRNA that will produce the correct polypeptide. Exons a and b are cotranscribed with a gene for a ribosomal protein (rps4), so an endonuclease must cut the RNA. The fate of the rps4 RNA has been omitted in the figure. The nad2 exons must be joined from two separate primary transcripts by both cis- and trans-splicing. In this gene, editing of the sequence at the RNA level changes 31 bases (symbolized by green dots) exclusively in exons. For clarity, the different RNA maturation processes are depicted here as consecutive events, whereas in vivo they appear to take place in an unordered sequence or simultaneously. (After Lippok et al. 1996 and Unseld et al. 1997.) (Click image to enlarge.)

In plant mitochondria, an additional RNA processing event takes places—RNA editing (Covello and Gray 1989; Gualberto et al. 1989; Hiesel et al. 1989). In this process, certain cytidines (C) are deaminated into uridines (U). Rarely, U is aminated to C instead. This means that the coding sequence is changed at the RNA level, in order to restore the correct information for translation where the DNA has diverged. Editing sites mainly occur in DNA encoding protein sequences and functional RNA, and are rare in noncoding mtDNA. In principle, the higher the importance of an individual base, the higher the probability that it constitutes an editing site. Despite the considerable amount of bases that can be edited (up to 15% of the bases of a gene), the functional relevance of this process and the consequent divergence in mtDNA sequence is not understood. Mitochondrial RNA editing has been found in land plants, but not in algae. It also occurs, though to a lesser extent, in plastids (Marchfelder et al. 1998).

The mitochondrial genome encodes only a small proportion of the mitochondrial proteins; less than 40 as compared to up to thousands of nuclear-encoded mitochondrial proteins (Emanuelsson et al. 2000). Through evolution, genes have been transferred from the mitochondrion to the nucleus. This is a process that still takes place, at least in plants. For example, subunit 2 of cytochrome oxidase is encoded by the mitochondrial genome in all investigated eukaryotes except for some legumes and protists. Within the legume family, several intermediates of the gene transfer have been found. For example, a functional gene can be found in the mitochondrial genome and an unexpressed one in the nuclear genome, or vise versa (Adams et al. 1999). In order for a mitochondrial gene to be successfully transferred to the nucleus, the protein-coding frame must be integrated into the chromosome, and several other modifications must be made (Figure 3). For example: (1) a promoter for nuclear transcription must be inserted at the beginning of the gene, (2) the mRNA should acquire sequences specifying the addition of a poly-A tail, and (3) a targeting sequence is necessary for the cytosolically synthesized protein to be imported into the mitochondrion. For proper function, elements regulating the expression in different cell types and under different conditions must also be included.

Figure 3 Transfer of a gene from the mitochondrion to the nucleus. A mitochondrial gene can be functionally transferred if several rare events take place. The mitochondrial mRNA must be reverse-transcribed and the resulting DNA integrated into a nuclear chromosome. DNA elements for expression of the gene and for directing the protein to the mitochondrion must then be assembled with it. If this takes place, the mitochondrial gene copy can be deleted without loss of function. (Click image to enlarge.)

Since the coding sequence for most mitochondrial genes in plants is edited at the RNA level, the mitochondrial genomic sequence will not code for a functional protein if inserted into the nucleus. Therefore, the DNA that is integrated into the nuclear genome must in most cases be made by reverse transcription from mitochondrial mRNA. Recently, upon sequencing the nuclear genome of Arabidopsis thaliana, a copy of most of the mtDNA was found integrated in a nuclear chromosome (Lin et al. 1999), indicating that large segments of DNA can move between cellular compartments. However, most of these mitochondrial genes transferred to the nuclear genome cannot produce functional proteins unless bases corresponding to editing sites are corrected by mutation. However, this is unlikely to take place before other mutations have further disrupted the sequence. As opposed to other mitochondrial genomes, plant mitochondria utilize the universal genetic code. Thus, this factor will not prevent or delay the transfer of genes between plant organelles as it may do in other eukaryotes.

The special features of the mitochondrial genetic system in plants also have consequences for the study of mitochondrial genes. Due to RNA editing and trans-splicing, a reading frame is more difficult to identify based only on genomic sequence. Also, protein sequences in databases are automatically translated from genomic DNA sequences without compensating for RNA editing in many cases. This protocol would not work for mitochondrial genes.

References

Adams, K. L., Song, K., Roessler, P. G., Nugent, J. M., Doyle, J. L., Doyle, J. J., and Palmer, J. D. (1999) Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes. Proc. Nat. Acad. Sci. 96: 13863–13868.

Backert, S., Nielsen, B. L., and Börner, T. (1997) The mystery of the rings: Structure and replication of mitochondrial genomes from higher plants. Trends Plant Sci. 2: 477–483.

Covello, P. S., and Gray, M. W. (1989) RNA editing in plant mitochondria. Nature 341: 662–666.

Gagliardi, D., and Leaver, C.J. (1999) Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower. EMBO J. 18:3757–3766.

Gray, M. W., Burger, G., and Lang, F. (1999) Mitochondrial evolution. Science 283: 1476–1481.

Gualberto, J. M., Lamattina, L., Bonnard, G., Weil, J.-H., and Grienenberger J.-M. (1989) RNA editing in wheat mitochondria results in the conservation of protein sequence. Nature 341: 660–662.

Hiesel, R., Wissinger, B., Schuster, W., and Brennicke, A. (1989) RNA editing in plant mitochondria. Science 246:1632–1634.

Kuhn, J., Tengler, U. and Binder, S. (2001) Transcript lifetime is balanced between stabilizing stem-loop structures and degradation-promoting polyadenylation in plant mitochondria. Mol. Cell. Biol. 21: 731–742.

Kühn, K., Weihe, A., and Börner, T. (2005) Multiple promoters are a common feature of mitochondrial genes in Arabidopsis. Nucleic Acids Res. 33: 337–346.

Lin, X., Kaul, S., Rounsley, S., Shea, T. P., Benito, M. I., et al. (1999) Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana. Nature 402: 761–768.

Lippok, B., Brennicke, A., and Unseld, M. (1996) The rps4-gene is encoded upstream of the nad2-gene in Arabidopsis mitochondria. Biol. Chem. Hoppe Seyler 377: 251–257.

Marchfelder, A., Binder, S., Brennicke, A., and Knoop, V. (1998) RNA editing by base conversion in plant organellar RNAs. In Modification and Editing of RNA, H. Grosjean, and R Benne, eds. ASM Press Washington, D.C.

Marienfeld, J., Unseld, M., and Brennicke, A. (1999) The mitochondrial genome of Arabidopsis is composed of both native and immigrant information. Trends. Plant Sci. 4: 495–502.

Millar, A.H., Heazlewood, J.L., Kristensen, B.K., Braun, H.P., and Møller, I.M. (2005) The plant mitochondrial proteome. Trends Plant Sci. 10: 36–43.

Sugiura, M., and Takeda, Y. (2000) Nucleic acids. In Biochemistry & Molecular Biology of Plants, B. B. Buchanan, W. Guissem, and R. L. Jones eds., pp. 676–728. American Society of Plant Physiologists, Rockville, MD.

Sugiyama, Y., Watase, Y., Nagase, M., Makita, N., Yagura, S., Hirai, A., and Sugiura, M. (2005) The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants. Mol. Genet. Genomics 272: 603–615.

Unseld, M., Marienfeld, J. R., Brandt, P., and Brennicke, A. (1997) The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature Genet. 15: 57–61.