The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana

Abstract

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

Figure 1

Gene organization of the rapeseed mitochondrial genome. Genes homologous to known protein-coding genes are indicated by red boxes. The blue boxes represent rRNA genes. Pink boxes represent unidentified ORFs longer than 150 amino acids. tRNA genes are represented by yellow boxes. Pseudo genes including plastid gene segments are shown in pale green. orf222, a cms-related gene (20), is shown by a green box. Arrowheads indicate the direction of reading frames. Dark green boxes located inside the circle represent 2 kb repeat regions. *From Heazlewood et al. (32); **From Sabar et al. (18).

Figure 2

Alignment of protein sequence deduced for rps14 of rapeseed mitochondria with other plant RPS14 protein sequences. Rapeseed, rapeseed rps14 (this study); Oenothera, deduced from Oenothera rps14 cDNA (26); Arabidopsis, deduced from Arabidopsis nuclear rps14 (28); Rice, deduced from rice nuclear rps14 (30); Marchantia, deduced from Marchantia mitochondrial rps14 (33). Amino acids that are conserved in at least three species are highlighted. Editing positions on Oenothera rps14 transcripts are indicated by triangles, and changed amino acids are shaded.

Figure 3

(A) Nucleotide sequence of the ccmC locus in rapeseed mitochondria. The polypeptide sequence is given below the genomic sequence. Codons altered by RNA editing are boxed together with the corresponding amino acid alterations. The edited nucleotides are shown in lower case letters. The ends of ccmC transcripts in Arabidopsis are boxed with double lines (S.Binder and P.Giegé, personal communications). The positions of oligonucleotides used for cDNA synthesis and PCR amplification are indicated by arrows. (B) Agarose gel electrophoresis of RT–PCR and PCR amplification products. The names of the oligonucleotides used as reverse primers are shown above the gel. Lane 1, PCR products using mitochondrial DNA as a template; lane 2, RT–PCR products with reverse transcriptase; lane 3, RT–PCR products without reverse transcriptase. M, DNA marker.