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The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNACys(GCA)

The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.)... © 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No. 13 2571–2576 The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel Cys gene for tRNA (GCA) Tomohiko Kubo, Satsuki Nishizawa, Akira Sugawara, Noriko Itchoda, Amy Estiati and Tetsuo Mikami* Laboratory of Genetic Engineering, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Received February 22, 2000; Revised and Accepted May 6, 2000 DDBJ/EMBL/GenBank accession nos AP000396, AP000397 ABSTRACT higher plant mtDNAs exhibit a number of unique structural features (e.g. large size, rapid evolutionary rearrangements and We determined the complete nucleotide sequence of chloroplast DNA insertions) and specific modes of expression the mitochondrial genome of an angiosperm, sugar (e.g. cis-and trans-splicing, RNA editing and universal genetic beet (Beta vulgaris cv TK81-O). The 368 799 bp code usage) (4). Moreover, they encode significantly more genome contains 29 protein, five rRNA and 25 tRNA genes than do their fungal and animal counterparts. Ribosomal genes, most of which are also shared by the mito- protein genes as well as the genes involved in cytochrome chondrial genome of Arabidopsis thaliana, the only c biogenesis comprise a significant fraction of such additional genes identified so far in plant mitochondria (4). The complete other completely sequenced angiosperm mitochon- sequence of the Arabidopsis mtDNA reveals approximately drial genome. However, four genes identified here 85 open reading frames (ORFs) of unknown function, some of (namely rps13, trnF-GAA, ccb577 and trnC2-GCA) are which may encode plant-specific mitochondrial proteins (3). missing in Arabidopsis mitochondria. In addition, It is also interesting to note that the study of different plant four genes found in Arabidopsis (ccb228, rpl2, rpl16 lineages has demonstrated the recent evolutionary transfer of and trnY2-GUA) are entirely absent in sugar beet or certain mitochondrial genes to the nucleus, and therefore the present only in severely truncated form. Introns, gene content of mitochondria may vary in different species (4). duplicated sequences, additional reading frames and The determination of the complete mtDNA sequence of inserted foreign sequences (chloroplast, nuclear and another angiosperm would thus allow us to examine the entire plasmid DNA sequences) contribute significantly to coding potential of plant mitochondrial genomes and to ascer- the overall size of the sugar beet mitochondrial tain whether or not the plant mitochondrial gene content and genome. Nevertheless, 55.6% of the genome has no organization have been conserved during flowering plant evolution. In addition, it should greatly facilitate the identification obvious features of information. We identified a Cys of new genes and provide insights into specific mechanisms novel tRNA gene (trnC2-GCA) which shows no Cys underlying mitochondrial genome divergence. sequence homology with any tRNA genes In this article we describe the complete sequence of the mito- reported so far in higher plants. Intriguingly, this chondrial genome of sugar beet (Beta vulgaris L.). We found tRNA gene is actually transcribed into a mature an intriguing diversity of gene complements between sugar Cys tRNA, whereas the native tRNA gene (trnC1-GCA) beet and Arabidopsis mitochondrial genomes, although most is most likely a pseudogene. of the genes reported in Arabidopsis are shared by sugar beet. Of particular interest is the occurrence in sugar beet mtDNA of Cys agenefor tRNA which shows no significant similarity to the INTRODUCTION Cys plant mitochondrial, chloroplast or nuclear tRNA genes Over the past decade much effort has been directed towards identified to date. This novel gene is actually transcribed in determining the entire genomic sequence of the mitochondrial Cys sugar beet mitochondria whereas the standard (native) tRNA DNAs (mtDNAs) from a wide array of organisms (1). gene seems not to be functional. However, the studied organisms are taxonomically highly biased; most of the complete mtDNA sequences published are MATERIALS AND METHODS from animals, protists and ascomycete fungi, whereas the available sequences for plants are limited to the liverwort Sequencing procedures Marchantia polymorpha (2) and the angiosperm Arabidopsis thaliana (3). The clone bank covering the entire mitochondrial genome of Despite the fact that mitochondria have essentially the same normal fertile sugar beet (cv TK81-O) was previously metabolic function in all eukaryotes which contain them, constructed using λ-DASH (Stratagene) and cosmid pHC79 *To whom correspondence should be addressed. Tel: +81 11 706 2806; Fax: +81 11 716 0879; Email: [email protected] 2572 Nucleic Acids Research, 2000, Vol. 28, No. 13 (Boehringer) (5). We selected three cosmid clones for number of editing events in sugar beet is somewhat fewer than sequencing the rrn26-carrying repeats and 21 λ clones for in Arabidopsis (441 editings). We also experimentally verified sequencing the remaining region of the genome. mtDNA in the RNA editing-mediated creation of initiation (nad1, nad4L each λ clone was amplified by long PCR using an LA-PCR kit and atp6) and termination codons (atp6 and atp9), which is not (Takara Shuzo) and then physically sheared. The resultant documented in the corresponding Arabidopsis transcripts fragments were blunt-ended using T4 DNA polymerase and (10,11; T.Kubo et al., unpublished data). Klenow fragment and subcloned into pBluescript (Stratagene). Repeated sequences ThepurifiedcosmidDNA wassonicated andusedfor subcloning. Shotgun sequencing was continued until a We previously reported that the sugar beet rrn26 gene is found minimum of 4-fold sequence coverage was achieved for each exclusively within a three-copy recombining-repeat family (the clone. The DNA sequencer used was a Li-COR 4200L (Li-COR). rrn26 repeat) (5). The present study indicates that the common The PCR products obtained by amplification of the sugar beet sequence unit shared by all three rrn26 repeat copies is 6222 bp mtDNA were used for gap filling and confirmation of the long and contains trnfM-CAU as well as rrn26 (Fig. 1). As can be sequences. Sequence alignments were generated using seen in Figure 1, in two of the three repeat copies sequence Sequencher 3.0 software (Gene Codes). A database search was identity continues a further 16 517 bp beyond the downstream done with the BLAST network service (http:// junction (93 bp downstream from trnfM-CAU) of the common www.dna.affrc.go.jp/htdocs/Blast/blast2.html ) and a tRNA sequence unit. The same relationship holds between two of the gene search with the tRNAscan-SE service (http:// three upstream rrn26 repeat flanks, which share an additional www.genetics.wustl.edu/eddy/tRNAscan-SE/ ) (6). The 3528 bp sequence beyond the upstream junction of the nucleotide sequence reported in this paper has been deposited common sequence unit (Fig. 1). We found perfect identity in in the DDBJ/GenBank/EMBL databases under accession nos the base sequence over the entire length of the repeated AP000396 and AP000397. sequence elements, suggesting that direct comparison of the repeat copies and correction of any mutational changes that RNA isolation and hybridization appear in any one of the copies are ongoing processes. Mitochondria were prepared from green leaves as described The sugar beet mtDNA also contains 72 short repeated DNA (7) and were used for RNA isolation (8). Aliquots of total segments, the sizes of which vary from 50 to 626 bp. These mtRNA (5 µ g) were electrophoresed through 1.4% agarose short repeats seem not to be active in frequent recombinations gels containing 0.66 M formaldehyde. Gels were blotted onto and they can be divided into 35 unrelated families. The Hybond N membranes (Amersham) and hybridized with a repeated sequence elements cover 10.3% of the genome. P-labeled probe. Promiscuous sequences It is well documented that plant mtDNAs contain many RESULTS AND DISCUSSION sequences of foreign origin. Chloroplast DNA (cpDNA) Conserved genes sequences provided the first such example (12) and sequences of nuclear origin are also found in plant mitochondrial Our strategy for analysis of the sugar beet mitochondrial genomes (13). Our analysis indicates that cpDNA-derived genome was based upon assembling the sequences of partially sequences comprise 2.1% of the mitochondrial genome of overlapping clones from λ and cosmid libraries and of long sugar beet. The cpDNA insertions vary in size from 25 to 3366 bp. PCR products which have been assigned on the physical map The largest fragment bears rps7, the last two exons of rps12 of the genome (5). The entire genome size of the sugar beet and trnV-GAC. Comparison of this 3.4 kb sequence to the mitochondrion is 368 799 bp, with an overall G+C content of corresponding sequences of sugar beet (accession no. 43.9%. Thesefigures arecomparabletothoseof Arabidopsis AB032426) and tobacco cpDNAs (Z00044) revealed that the (366 924 bp in size and a G+C content of 44.8%) (3). integrated sequence in question is closer to the sugar beet Homology searches to match the entire genomic sequence with cpDNA than to tobacco cpDNA (details to be published known sequences in the public DNA database detected elsewhere). This result supports the suggestion that cpDNA 29 protein coding genes, five rRNA genes and 25 tRNA genes, insertion took place after the divergence of sugar beet and which altogether account for 11.3% of the genome (Table 1). tobacco. The cpDNA insertions include six intact and most The positions of these genes in the sugar beet mitochondrial likely functional tRNA genes which are also shared by genome are depicted in Figure 1. Although most of the genes Arabidopsis mtDNA (Table 1). However, most of the remaining identified here are also found in Arabidopsis mitochondria, a cpDNA-like sequences are not detected in Arabidopsis. few differences are noted in the gene content between sugar beet and Arabidopsis. As shown in Table 1, the rps13, ccb577, We also found nuclear-like sequences covering 3.3% of the trnF-GAA and trnC2-GCA (see below) genes are present in mitochondrial genome of sugar beet. We identified 21 retro- sugar beet mitochondria but not in the mtDNA of Arabidopsis. transposon-like sequences with lengths of between 25 and Conversely, sugar beet mitochondria apparently lack four 2827 bp, only two of which exhibit similarity to the mitochondrial genes (ccb228, rpl2, rpl16 and trnY2-GUA) which are encoded retrotransposon sequences of Arabidopsis. The most conspicuous by Arabidopsis mtDNA. example is an orf84–orf100–orf78–orf764 cluster which seems An alignment of all known mitochondrial RNA editing sites to be related to the gypsy-type retrotransposon. This cluster (see for example 9) with the homologous nucleotide sequence hybridized to sugar beet nuclear DNA (data not shown) but no of sugar beet mtDNA allowed us to predict at least 370 C→U corresponding sequence was observed in Arabidopsis mito- editing sites in 28 gene/ORF transcripts of sugar beet. This chondria. Another noteworthy sequence of nuclear origin is a Nucleic Acids Research, 2000, Vol. 28, No. 13 2573 Table 1. Gene content of sugar beet (Bv) mtDNA compared to Arabidopsis (At) Gene (map co-ordinate in kb) Bv At Gene (map co-ordinate in kb) Bv At Complex I Ribosomal RNAs nad1* (102, 105, 152, 243) + + rrn5 (308) + + nad2* (75, 240) + + rrn18 (309) + + nad3 (263) + + rrn26 (125,212,348) + + nad4 (285) + + Intronic ORF (in nad1-i4) nad4L (55) + + mat-r (244) + + nad5* (141, 171, 319) + + Sec-independent membrane targeting ++ nad6 (193) + + and translocation system nad7 (216) + + tatC (199) + + nad9 (0) + + Other ORF Complex II orf25 (54) + + sdh4 – ψ?tRNAs Complex III ‘Native’ cob (179) + + trnC1-GCA (83) ψ + Complex IV trnE-UUC (91) + + cox1 (238) + + trnF-GAA (329) + – cox2 (234) + + trnG-GCC (315) + + cox3 (297) + + trnI-CAU (197) + + Complex V trnK-UUU (163) + + atp1 (337) + + trnfM-CAU (125, 153, 216, 352) + + atp6 (201) + + trnP-UGG (329) + + atp8 (299) + + trnQ-UUG (313) + + atp9 (72) + + trnS-GCU (332) + + Cytochrome c biogenesis trnS-UGA (154) + + ccb206 (156) + + trnY1-GUA (80) + + ccb228 (49) ψ + ‘Chloroplast-like’ ccb438 (37) + + trnD-GUC (289) + + ccb577 (33) + ψ? trnH-GUG (93) + + Ribosomal proteins trnN-GUU (81) + + rpl2 –+ trnM-CAU (89) + + rpl5 (181) + + trnP-UGG (1) ψψ rpl16 –+ trnS-GGA (250) + + rps3 (271) + + trnV-GAC (7) ψ – rps4 (191) + + trnW-CCA (1) + + rps7 (231) + + Converted from trnF-GAA rps12 (263) + + trnY2-GUA – + rps13 (107) + – Origin unknown trnC2-GCA (123, 354) + – *, trans-splicing; +, present; ψ, pseudogene; ψ?, likely pseudogene; –, absent; a, three copies; b, four copies; c, two copies. 168 bp segment with homology to (R)-mandelonitrile lyase which are categorized as group II introns based on their –21 (P =8 × 10 ). distinctive secondary structures. The sum of all intron lengths is ~25 800 bp, representing 7.0% of the genome. A total of six Introns trans-splicing introns are found in the nad1, nad2 and nad5 As in other higher plants, the sugar beet mitochondrial rRNA genes and 14 cis-splicing introns are found in the seven protein coding genes (cox2, nad1, nad2, nad4, nad5, nad7 and and tRNA genes lack introns. Nonetheless, sugar beet mtDNA has at least 20 introns in seven protein coding genes, all of ccb438). Interestingly, we found sugar beet rps3 to completely 2574 Nucleic Acids Research, 2000, Vol. 28, No. 13 Figure 1. Gene map of the sugar beet mitochondrial genome. Protein, rRNA and tRNA coding genes and ORFs longer than 100 amino acids are indicated by gray boxes. cis-splicing introns are shown by thin lines. Orientations are clockwise for the genes shown outside the circle and counterclockwise for the genes shown inside the circle. Abbreviations for the three classes of tRNA genes are: na, native (genuine); cp, chloroplast-like; un, origin unknown. Three-copyrepeated sequences which are active in recombination are indicated by bold lines. Map coordinate 0 has been arbitrarily assigned to the first nucleotide of the nad9 ORF (top of the figure) and numbering proceeds clockwise. lack introns. This contrasts with the situation reported in other important protein information. Of the sugar beet mitochondrial plant species (3,14,15), where the rps3 coding region is inter- genome sequence 3.6% is homologous to mitochondrial rupted by a single intron. We also noted that the sugar beet plasmid DNAs. These sequences include segments of DNA nad4 gene does not contain the second intron found in several and RNA polymerase-related ORFs (accession no. Y10854). higher plants, including both monocots and dicots (16; N.Itchoda, We also found sequences with homology to the 5′-and 3′- T.Kubo and T.Mikami, manuscript in preparation). untranslated regions and the intergenic sequences of mitochon- drial genes in other plant species; they do not exceed 6.8% of Residual sequences the sugar beet genome. The major portion (55.6%) of the We searched for ORFs longer than 60 codons that begin with a genome has no similarity to any nucleotide or protein methionine codon and end with a classical termination codon. sequences in the public databases. A total of 559 ORFs were identified (93 ORFs are longer than tRNA genes 100 codons in size), none of which, however, exhibited apparent homology to any reported genes (or to any of the Twenty-five tRNA genes were identified by homology to their Arabidopsis ORFs; 3) or carried significant protein motifs. It respective counterparts in Arabidopsis and other higher plants thus seems unlikely that these ORFs code for additional and by their predicted cloverleaf structures, which define Nucleic Acids Research, 2000, Vol. 28, No. 13 2575 Figure 3. Northern blot analysis to detect and determine the relative sizes of Cys RNA transcripts of the two sugar beet tRNA genes. Hybridization probes were trnC2-GCA-, trnC1-GCA-, trnN-GUU- and trnY1-GUA-specific sequences whose positions are given in Figure 1. The sizes of the transcripts are shown in kilobases. gene (Fig. 3). In addition, a weaker signal (7.0 kb) indicated the presence of larger precursor molecules in the analyzed RNA fraction (Fig. 3). A probe that contains the the 5′-flanking Figure 2. Cloverleaf structure deduced from the trnC1-GCA and trnC2-GCA region of the upstream trnfM-CAU gene (Fig. 1) hybridized with gene sequences in sugar beet mitochondria. A mutation (T54→G54) in trnC1- the 7.0 kb transcript (data not shown), an observation that is GCA is indicatedbyanarrow. consistent with the co-transcription of trnfM-CAU and trnC2- GCA. In contrast, a trnC1-GCA-specific probe hybridized only to an RNA of 3.4 kb, indicating that the trnC1-GCA transcript unambiguous anticodons (Table 1). As shown in Figure 1, remains linked to a transcript that includes the downstream trnfM-CAU and trnC2-GCA are present in four and two trnN-GUU and trnY1-GUA genes (Fig. 1). This was confirmed copies, respectively, whereas the other tRNA genes occur by hybridization experiments with the trnN-GUU and trnY1-GUA singly. We previously reported that chloroplast-like trnP-UGG probes, which identified a 3.4 kb transcript as well as tRNA- is not transcribed in sugar beet mitochondria (17). The same sized molecules (Fig. 3). These results lead us to suppose that appears to hold true for chloroplast-like trnV-GAC (data not although the trnC1-GCA sequence is transcribed, it is rarely or shown). As there are no mitochondrially encoded tRNA species never processed into a mature molecule. Since no other for five amino acids (alanine, arginine, leucine, threonine and Cys sequence encoding tRNA is foundinthe sugar beet mito- valine) in sugar beet, tRNA import from the cytosol must be chondrial genome, it seems reasonable to conclude that the invoked as the mechanism for making up the deficit. Cys trnC2-GCA identified here is the only functional tRNA gene Our analysis also indicates the presence of two trnC-GCA in the sugar beet mitochondrial genome. genes; the first one is a native trnC-GCA (designated trnC1- GCA) and the other is a novel one (trnC2-GCA). When trnC1- CONCLUSIONS GCA was folded into a cloverleaf secondary structure, the invariant T54 was found to be replaced by a G (Fig. 2). T54 is We found that duplicated sequences, introns, additional genes believed to pair with A58 to yield the familiar L-shaped and inserted cpDNA sequences contribute significantly to the tertiary structure. Hence, a G54-A58 mismatch may have a overall size of the mitochondrial genome in sugar beet. It was deleterious effect on expression of the trnC1-GCA gene (see also noted that a large fraction (55.6%) of the sugar beet mito- below). On the other hand, the tRNA encoded by trnC2-GCA chondrial genomic sequences has no recognizable function and retains the invariant and semi-invariant nucleotides character- shows no apparent similarity to any sequence in the present istic of standard tRNA (18; Fig. 2). Surprisingly, the best databases. Furthermore, when the sugar beet mtDNA match between sugar beet trnC2-GCA and sequences in the sequences are compared to those of Arabidopsis, the sequences public databases is with the trnC-GCA from a Gram-negative shared by the two plant species total 78 057 bp, representing bacterium, Helicobacter pylori (accession no. AE000604), 21.2% of the sugar beet genome. Provided that many of the although it is too soon to say whether the homology (76%) is of enigmatic sequences in question are of nuclear origin, one can significance. It should also be noted that the trnC2-GCA- infer that many DNA transfer events have occurred separately specific probe failed to hybridize with cpDNA and nuclear in the two evolutionary lineages leading to sugar beet and DNA from sugar beet (data not shown). Arabidopsis, respectively. A further notable finding is that the sugar beet mtDNA The native trnC is likely a pseudogene whereas the novel Cys contains a novel tRNA gene (trnC2-GCA) which shows no trnC is expressed sequence homology to either the native or chloroplast-like Cys Northern blot analysis was carried out to test for the presence tRNA genes identified so far in plant mitochondria. The Cys in sugar beet mitochondria of RNAs corresponding to the trnC2-GCA is most likely the only functional tRNA gene in trnC1-GCA and trnC2-GCA sequences, respectively. tRNA- the sugar beet mitochondrial genome, although it remains to be Cys sized molecules (~80 bp) were detected by hybridization with seen whether the nuclear DNA-encoded tRNA species is a trnC2-GCA-specific probe, confirming transcription of this imported from the cytoplasm into mitochondria in sugar beet. 2576 Nucleic Acids Research, 2000, Vol. 28, No. 13 We have recently cloned a sugar beet cDNA encoding REFERENCES cysteinyl-tRNA synthase. Interestingly, this protein has been 1. Gray,M.W., Lang,B.F., Cedergren,R., Golding,G.B., Lemieux,C., foundtobeimportedinto mitochondria (T.Kubo et al., unpub- Sankoff,D., Turmel,M., Brossard,N., Delage,E., Littlejohn,T.G. et al. lished data). Whether the enzyme efficiently recognizes and (1998) Nucleic Acids Res., 26, 865–878. aminoacylates the trnC2-GCA transcript is currently under 2. Oda,K., Yamato,K., Ohta,E., Nakamura,Y., Takemura,M., Nozato,N., investigation. Preliminary PCR surveys of 21 diverse genera of Akashi,K., Kanegae,T., Ogura,Y., Kohchi,T. and Ohyama,K. (1992) J. Mol. Biol., 223,1–7. angiosperms indicate that the trnC2-GCA homolog is present 3. Unseld,M., Marienfeld,J.R., Brandt,P. and Brennicke,A. (1997) only in members of Caryophyllales (T.Kubo et al., unpub- Nature Genet., 15, 57–61. lished data). This raises the possibility that Caryophyllales 4. Schuster,W. and Brennicke,A. (1994) Annu. Rev. Plant Physiol. Plant acquired the trnC2-GCA sequence by horizontal transfer Mol. Biol., 45, 61–78. events from a distantly related organism. Most recently, Cho 5. Kubo,T., Satoh,Y., Muro,T., Kinoshita,T. and Mikami,T. (1995) et al. (19) have described the horizontal transfer of a group I Curr. Genet., 28, 235–241. intron from a fungal donor to the mitochondrial genome of 6. Lowe,T. and Eddy,S.R. (1997) Nucleic Acids Res., 25, 955–964. vascular plants. An alternative possibility that should be 7. Mikami,T., Kishima,Y., Sugiura,M. and Kinoshita,T. (1985) Theor. Appl. considered is that trnC2-GCA resulted from extensive Genet., 71, 166–171. genomic rearrangements, as exemplified in the trnY-GUA 8. Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156–159. 9. Giege,P. and Brennicke,A. (1999) Proc. Natl Acad. Sci. USA, 96, converted from trnF-GAA in Arabidopsis mitochondria (20) 15324–15329. or chimeric ORFs responsible for cytoplasmic male sterility in 10. Kubo,T., Yamamoto,M.P. and Mikami,T. (2000) Theor. Appl. Genet., various plants (21). Further studies are necessary to fully 100, 214–220. understand the origin and evolution of trnC2-GCA. 11. Onodera,Y., Yamamoto,M.P., Kubo,T. and Mikami,T. (1999) J. Plant Physiol., 155, 656–660. 12. Schuster,W. and Brennicke,A. (1988) Plant Sci., 54, 1–10. ACKNOWLEDGEMENTS 13. Knoop,V., Unseld,M., Marienfeld,J., Brandt,P., Sunkel,S., Ullrich,H. and We wish to thank Drs S. Tabata, T. Kaneko, Y. Nakamura and Brennicke,A. (1996) Genetics, 142, 579–585. K. Akashi for valuable discussions and suggestions, Dr T. 14. Hunt,M.D. and Newton,K.J. (1991) EMBO J., 10, 1045–1052. 15. Schuster,W. and Brennicke,A. (1991) Nucleic Acids Res., 19, 6923–6928. Tsudzuki for figure drawing and Drs Y. Kishima and Y. 16. Gass,D.A., Makaroff,C.A. and Palmer,J.D. (1992) Curr. Genet., 21, 423–430. Nishita for technical advice and continuous encouragement. 17. Kubo,T., Yanai,Y., Kinoshita,T. and Mikami,T. (1995) Curr. Genet., 27, This work was done in part at the Research Center for 285–289. Molecular Genetics, Hokkaido University, and was supported 18. McClain,W.H. (1993) J. Mol. Biol., 234, 257–280. in part by Grants in Aid for Scientific Research from the 19. Cho,Y., Qiu,Y.-L. and Palmer,J.D. (1998) Proc. Natl Acad. Sci. USA, 95, Ministry of Education, Science and Culture, Japan, and a grant 14244–14249. from the Program for Promotion of Basic Research Activities 20. Chen,H.C., Wintz,H., Weil,J.H. and Pillay,T.N. (1989) Nucleic Acids Res., for Innovative Biosciences, Japan. A.E. is the recipient of a 17, 2613–2621. Japanese Government (Monbusho) Scholarship. 21. Levings,C.S. (1993) Plant Cell, 5, 1285–1290. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNACys(GCA)

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© 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No. 13 2571–2576 The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel Cys gene for tRNA (GCA) Tomohiko Kubo, Satsuki Nishizawa, Akira Sugawara, Noriko Itchoda, Amy Estiati and Tetsuo Mikami* Laboratory of Genetic Engineering, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Received February 22, 2000; Revised and Accepted May 6, 2000 DDBJ/EMBL/GenBank accession nos AP000396, AP000397 ABSTRACT higher plant mtDNAs exhibit a number of unique structural features (e.g. large size, rapid evolutionary rearrangements and We determined the complete nucleotide sequence of chloroplast DNA insertions) and specific modes of expression the mitochondrial genome of an angiosperm, sugar (e.g. cis-and trans-splicing, RNA editing and universal genetic beet (Beta vulgaris cv TK81-O). The 368 799 bp code usage) (4). Moreover, they encode significantly more genome contains 29 protein, five rRNA and 25 tRNA genes than do their fungal and animal counterparts. Ribosomal genes, most of which are also shared by the mito- protein genes as well as the genes involved in cytochrome chondrial genome of Arabidopsis thaliana, the only c biogenesis comprise a significant fraction of such additional genes identified so far in plant mitochondria (4). The complete other completely sequenced angiosperm mitochon- sequence of the Arabidopsis mtDNA reveals approximately drial genome. However, four genes identified here 85 open reading frames (ORFs) of unknown function, some of (namely rps13, trnF-GAA, ccb577 and trnC2-GCA) are which may encode plant-specific mitochondrial proteins (3). missing in Arabidopsis mitochondria. In addition, It is also interesting to note that the study of different plant four genes found in Arabidopsis (ccb228, rpl2, rpl16 lineages has demonstrated the recent evolutionary transfer of and trnY2-GUA) are entirely absent in sugar beet or certain mitochondrial genes to the nucleus, and therefore the present only in severely truncated form. Introns, gene content of mitochondria may vary in different species (4). duplicated sequences, additional reading frames and The determination of the complete mtDNA sequence of inserted foreign sequences (chloroplast, nuclear and another angiosperm would thus allow us to examine the entire plasmid DNA sequences) contribute significantly to coding potential of plant mitochondrial genomes and to ascer- the overall size of the sugar beet mitochondrial tain whether or not the plant mitochondrial gene content and genome. Nevertheless, 55.6% of the genome has no organization have been conserved during flowering plant evolution. In addition, it should greatly facilitate the identification obvious features of information. We identified a Cys of new genes and provide insights into specific mechanisms novel tRNA gene (trnC2-GCA) which shows no Cys underlying mitochondrial genome divergence. sequence homology with any tRNA genes In this article we describe the complete sequence of the mito- reported so far in higher plants. Intriguingly, this chondrial genome of sugar beet (Beta vulgaris L.). We found tRNA gene is actually transcribed into a mature an intriguing diversity of gene complements between sugar Cys tRNA, whereas the native tRNA gene (trnC1-GCA) beet and Arabidopsis mitochondrial genomes, although most is most likely a pseudogene. of the genes reported in Arabidopsis are shared by sugar beet. Of particular interest is the occurrence in sugar beet mtDNA of Cys agenefor tRNA which shows no significant similarity to the INTRODUCTION Cys plant mitochondrial, chloroplast or nuclear tRNA genes Over the past decade much effort has been directed towards identified to date. This novel gene is actually transcribed in determining the entire genomic sequence of the mitochondrial Cys sugar beet mitochondria whereas the standard (native) tRNA DNAs (mtDNAs) from a wide array of organisms (1). gene seems not to be functional. However, the studied organisms are taxonomically highly biased; most of the complete mtDNA sequences published are MATERIALS AND METHODS from animals, protists and ascomycete fungi, whereas the available sequences for plants are limited to the liverwort Sequencing procedures Marchantia polymorpha (2) and the angiosperm Arabidopsis thaliana (3). The clone bank covering the entire mitochondrial genome of Despite the fact that mitochondria have essentially the same normal fertile sugar beet (cv TK81-O) was previously metabolic function in all eukaryotes which contain them, constructed using λ-DASH (Stratagene) and cosmid pHC79 *To whom correspondence should be addressed. Tel: +81 11 706 2806; Fax: +81 11 716 0879; Email: [email protected] 2572 Nucleic Acids Research, 2000, Vol. 28, No. 13 (Boehringer) (5). We selected three cosmid clones for number of editing events in sugar beet is somewhat fewer than sequencing the rrn26-carrying repeats and 21 λ clones for in Arabidopsis (441 editings). We also experimentally verified sequencing the remaining region of the genome. mtDNA in the RNA editing-mediated creation of initiation (nad1, nad4L each λ clone was amplified by long PCR using an LA-PCR kit and atp6) and termination codons (atp6 and atp9), which is not (Takara Shuzo) and then physically sheared. The resultant documented in the corresponding Arabidopsis transcripts fragments were blunt-ended using T4 DNA polymerase and (10,11; T.Kubo et al., unpublished data). Klenow fragment and subcloned into pBluescript (Stratagene). Repeated sequences ThepurifiedcosmidDNA wassonicated andusedfor subcloning. Shotgun sequencing was continued until a We previously reported that the sugar beet rrn26 gene is found minimum of 4-fold sequence coverage was achieved for each exclusively within a three-copy recombining-repeat family (the clone. The DNA sequencer used was a Li-COR 4200L (Li-COR). rrn26 repeat) (5). The present study indicates that the common The PCR products obtained by amplification of the sugar beet sequence unit shared by all three rrn26 repeat copies is 6222 bp mtDNA were used for gap filling and confirmation of the long and contains trnfM-CAU as well as rrn26 (Fig. 1). As can be sequences. Sequence alignments were generated using seen in Figure 1, in two of the three repeat copies sequence Sequencher 3.0 software (Gene Codes). A database search was identity continues a further 16 517 bp beyond the downstream done with the BLAST network service (http:// junction (93 bp downstream from trnfM-CAU) of the common www.dna.affrc.go.jp/htdocs/Blast/blast2.html ) and a tRNA sequence unit. The same relationship holds between two of the gene search with the tRNAscan-SE service (http:// three upstream rrn26 repeat flanks, which share an additional www.genetics.wustl.edu/eddy/tRNAscan-SE/ ) (6). The 3528 bp sequence beyond the upstream junction of the nucleotide sequence reported in this paper has been deposited common sequence unit (Fig. 1). We found perfect identity in in the DDBJ/GenBank/EMBL databases under accession nos the base sequence over the entire length of the repeated AP000396 and AP000397. sequence elements, suggesting that direct comparison of the repeat copies and correction of any mutational changes that RNA isolation and hybridization appear in any one of the copies are ongoing processes. Mitochondria were prepared from green leaves as described The sugar beet mtDNA also contains 72 short repeated DNA (7) and were used for RNA isolation (8). Aliquots of total segments, the sizes of which vary from 50 to 626 bp. These mtRNA (5 µ g) were electrophoresed through 1.4% agarose short repeats seem not to be active in frequent recombinations gels containing 0.66 M formaldehyde. Gels were blotted onto and they can be divided into 35 unrelated families. The Hybond N membranes (Amersham) and hybridized with a repeated sequence elements cover 10.3% of the genome. P-labeled probe. Promiscuous sequences It is well documented that plant mtDNAs contain many RESULTS AND DISCUSSION sequences of foreign origin. Chloroplast DNA (cpDNA) Conserved genes sequences provided the first such example (12) and sequences of nuclear origin are also found in plant mitochondrial Our strategy for analysis of the sugar beet mitochondrial genomes (13). Our analysis indicates that cpDNA-derived genome was based upon assembling the sequences of partially sequences comprise 2.1% of the mitochondrial genome of overlapping clones from λ and cosmid libraries and of long sugar beet. The cpDNA insertions vary in size from 25 to 3366 bp. PCR products which have been assigned on the physical map The largest fragment bears rps7, the last two exons of rps12 of the genome (5). The entire genome size of the sugar beet and trnV-GAC. Comparison of this 3.4 kb sequence to the mitochondrion is 368 799 bp, with an overall G+C content of corresponding sequences of sugar beet (accession no. 43.9%. Thesefigures arecomparabletothoseof Arabidopsis AB032426) and tobacco cpDNAs (Z00044) revealed that the (366 924 bp in size and a G+C content of 44.8%) (3). integrated sequence in question is closer to the sugar beet Homology searches to match the entire genomic sequence with cpDNA than to tobacco cpDNA (details to be published known sequences in the public DNA database detected elsewhere). This result supports the suggestion that cpDNA 29 protein coding genes, five rRNA genes and 25 tRNA genes, insertion took place after the divergence of sugar beet and which altogether account for 11.3% of the genome (Table 1). tobacco. The cpDNA insertions include six intact and most The positions of these genes in the sugar beet mitochondrial likely functional tRNA genes which are also shared by genome are depicted in Figure 1. Although most of the genes Arabidopsis mtDNA (Table 1). However, most of the remaining identified here are also found in Arabidopsis mitochondria, a cpDNA-like sequences are not detected in Arabidopsis. few differences are noted in the gene content between sugar beet and Arabidopsis. As shown in Table 1, the rps13, ccb577, We also found nuclear-like sequences covering 3.3% of the trnF-GAA and trnC2-GCA (see below) genes are present in mitochondrial genome of sugar beet. We identified 21 retro- sugar beet mitochondria but not in the mtDNA of Arabidopsis. transposon-like sequences with lengths of between 25 and Conversely, sugar beet mitochondria apparently lack four 2827 bp, only two of which exhibit similarity to the mitochondrial genes (ccb228, rpl2, rpl16 and trnY2-GUA) which are encoded retrotransposon sequences of Arabidopsis. The most conspicuous by Arabidopsis mtDNA. example is an orf84–orf100–orf78–orf764 cluster which seems An alignment of all known mitochondrial RNA editing sites to be related to the gypsy-type retrotransposon. This cluster (see for example 9) with the homologous nucleotide sequence hybridized to sugar beet nuclear DNA (data not shown) but no of sugar beet mtDNA allowed us to predict at least 370 C→U corresponding sequence was observed in Arabidopsis mito- editing sites in 28 gene/ORF transcripts of sugar beet. This chondria. Another noteworthy sequence of nuclear origin is a Nucleic Acids Research, 2000, Vol. 28, No. 13 2573 Table 1. Gene content of sugar beet (Bv) mtDNA compared to Arabidopsis (At) Gene (map co-ordinate in kb) Bv At Gene (map co-ordinate in kb) Bv At Complex I Ribosomal RNAs nad1* (102, 105, 152, 243) + + rrn5 (308) + + nad2* (75, 240) + + rrn18 (309) + + nad3 (263) + + rrn26 (125,212,348) + + nad4 (285) + + Intronic ORF (in nad1-i4) nad4L (55) + + mat-r (244) + + nad5* (141, 171, 319) + + Sec-independent membrane targeting ++ nad6 (193) + + and translocation system nad7 (216) + + tatC (199) + + nad9 (0) + + Other ORF Complex II orf25 (54) + + sdh4 – ψ?tRNAs Complex III ‘Native’ cob (179) + + trnC1-GCA (83) ψ + Complex IV trnE-UUC (91) + + cox1 (238) + + trnF-GAA (329) + – cox2 (234) + + trnG-GCC (315) + + cox3 (297) + + trnI-CAU (197) + + Complex V trnK-UUU (163) + + atp1 (337) + + trnfM-CAU (125, 153, 216, 352) + + atp6 (201) + + trnP-UGG (329) + + atp8 (299) + + trnQ-UUG (313) + + atp9 (72) + + trnS-GCU (332) + + Cytochrome c biogenesis trnS-UGA (154) + + ccb206 (156) + + trnY1-GUA (80) + + ccb228 (49) ψ + ‘Chloroplast-like’ ccb438 (37) + + trnD-GUC (289) + + ccb577 (33) + ψ? trnH-GUG (93) + + Ribosomal proteins trnN-GUU (81) + + rpl2 –+ trnM-CAU (89) + + rpl5 (181) + + trnP-UGG (1) ψψ rpl16 –+ trnS-GGA (250) + + rps3 (271) + + trnV-GAC (7) ψ – rps4 (191) + + trnW-CCA (1) + + rps7 (231) + + Converted from trnF-GAA rps12 (263) + + trnY2-GUA – + rps13 (107) + – Origin unknown trnC2-GCA (123, 354) + – *, trans-splicing; +, present; ψ, pseudogene; ψ?, likely pseudogene; –, absent; a, three copies; b, four copies; c, two copies. 168 bp segment with homology to (R)-mandelonitrile lyase which are categorized as group II introns based on their –21 (P =8 × 10 ). distinctive secondary structures. The sum of all intron lengths is ~25 800 bp, representing 7.0% of the genome. A total of six Introns trans-splicing introns are found in the nad1, nad2 and nad5 As in other higher plants, the sugar beet mitochondrial rRNA genes and 14 cis-splicing introns are found in the seven protein coding genes (cox2, nad1, nad2, nad4, nad5, nad7 and and tRNA genes lack introns. Nonetheless, sugar beet mtDNA has at least 20 introns in seven protein coding genes, all of ccb438). Interestingly, we found sugar beet rps3 to completely 2574 Nucleic Acids Research, 2000, Vol. 28, No. 13 Figure 1. Gene map of the sugar beet mitochondrial genome. Protein, rRNA and tRNA coding genes and ORFs longer than 100 amino acids are indicated by gray boxes. cis-splicing introns are shown by thin lines. Orientations are clockwise for the genes shown outside the circle and counterclockwise for the genes shown inside the circle. Abbreviations for the three classes of tRNA genes are: na, native (genuine); cp, chloroplast-like; un, origin unknown. Three-copyrepeated sequences which are active in recombination are indicated by bold lines. Map coordinate 0 has been arbitrarily assigned to the first nucleotide of the nad9 ORF (top of the figure) and numbering proceeds clockwise. lack introns. This contrasts with the situation reported in other important protein information. Of the sugar beet mitochondrial plant species (3,14,15), where the rps3 coding region is inter- genome sequence 3.6% is homologous to mitochondrial rupted by a single intron. We also noted that the sugar beet plasmid DNAs. These sequences include segments of DNA nad4 gene does not contain the second intron found in several and RNA polymerase-related ORFs (accession no. Y10854). higher plants, including both monocots and dicots (16; N.Itchoda, We also found sequences with homology to the 5′-and 3′- T.Kubo and T.Mikami, manuscript in preparation). untranslated regions and the intergenic sequences of mitochon- drial genes in other plant species; they do not exceed 6.8% of Residual sequences the sugar beet genome. The major portion (55.6%) of the We searched for ORFs longer than 60 codons that begin with a genome has no similarity to any nucleotide or protein methionine codon and end with a classical termination codon. sequences in the public databases. A total of 559 ORFs were identified (93 ORFs are longer than tRNA genes 100 codons in size), none of which, however, exhibited apparent homology to any reported genes (or to any of the Twenty-five tRNA genes were identified by homology to their Arabidopsis ORFs; 3) or carried significant protein motifs. It respective counterparts in Arabidopsis and other higher plants thus seems unlikely that these ORFs code for additional and by their predicted cloverleaf structures, which define Nucleic Acids Research, 2000, Vol. 28, No. 13 2575 Figure 3. Northern blot analysis to detect and determine the relative sizes of Cys RNA transcripts of the two sugar beet tRNA genes. Hybridization probes were trnC2-GCA-, trnC1-GCA-, trnN-GUU- and trnY1-GUA-specific sequences whose positions are given in Figure 1. The sizes of the transcripts are shown in kilobases. gene (Fig. 3). In addition, a weaker signal (7.0 kb) indicated the presence of larger precursor molecules in the analyzed RNA fraction (Fig. 3). A probe that contains the the 5′-flanking Figure 2. Cloverleaf structure deduced from the trnC1-GCA and trnC2-GCA region of the upstream trnfM-CAU gene (Fig. 1) hybridized with gene sequences in sugar beet mitochondria. A mutation (T54→G54) in trnC1- the 7.0 kb transcript (data not shown), an observation that is GCA is indicatedbyanarrow. consistent with the co-transcription of trnfM-CAU and trnC2- GCA. In contrast, a trnC1-GCA-specific probe hybridized only to an RNA of 3.4 kb, indicating that the trnC1-GCA transcript unambiguous anticodons (Table 1). As shown in Figure 1, remains linked to a transcript that includes the downstream trnfM-CAU and trnC2-GCA are present in four and two trnN-GUU and trnY1-GUA genes (Fig. 1). This was confirmed copies, respectively, whereas the other tRNA genes occur by hybridization experiments with the trnN-GUU and trnY1-GUA singly. We previously reported that chloroplast-like trnP-UGG probes, which identified a 3.4 kb transcript as well as tRNA- is not transcribed in sugar beet mitochondria (17). The same sized molecules (Fig. 3). These results lead us to suppose that appears to hold true for chloroplast-like trnV-GAC (data not although the trnC1-GCA sequence is transcribed, it is rarely or shown). As there are no mitochondrially encoded tRNA species never processed into a mature molecule. Since no other for five amino acids (alanine, arginine, leucine, threonine and Cys sequence encoding tRNA is foundinthe sugar beet mito- valine) in sugar beet, tRNA import from the cytosol must be chondrial genome, it seems reasonable to conclude that the invoked as the mechanism for making up the deficit. Cys trnC2-GCA identified here is the only functional tRNA gene Our analysis also indicates the presence of two trnC-GCA in the sugar beet mitochondrial genome. genes; the first one is a native trnC-GCA (designated trnC1- GCA) and the other is a novel one (trnC2-GCA). When trnC1- CONCLUSIONS GCA was folded into a cloverleaf secondary structure, the invariant T54 was found to be replaced by a G (Fig. 2). T54 is We found that duplicated sequences, introns, additional genes believed to pair with A58 to yield the familiar L-shaped and inserted cpDNA sequences contribute significantly to the tertiary structure. Hence, a G54-A58 mismatch may have a overall size of the mitochondrial genome in sugar beet. It was deleterious effect on expression of the trnC1-GCA gene (see also noted that a large fraction (55.6%) of the sugar beet mito- below). On the other hand, the tRNA encoded by trnC2-GCA chondrial genomic sequences has no recognizable function and retains the invariant and semi-invariant nucleotides character- shows no apparent similarity to any sequence in the present istic of standard tRNA (18; Fig. 2). Surprisingly, the best databases. Furthermore, when the sugar beet mtDNA match between sugar beet trnC2-GCA and sequences in the sequences are compared to those of Arabidopsis, the sequences public databases is with the trnC-GCA from a Gram-negative shared by the two plant species total 78 057 bp, representing bacterium, Helicobacter pylori (accession no. AE000604), 21.2% of the sugar beet genome. Provided that many of the although it is too soon to say whether the homology (76%) is of enigmatic sequences in question are of nuclear origin, one can significance. It should also be noted that the trnC2-GCA- infer that many DNA transfer events have occurred separately specific probe failed to hybridize with cpDNA and nuclear in the two evolutionary lineages leading to sugar beet and DNA from sugar beet (data not shown). Arabidopsis, respectively. A further notable finding is that the sugar beet mtDNA The native trnC is likely a pseudogene whereas the novel Cys contains a novel tRNA gene (trnC2-GCA) which shows no trnC is expressed sequence homology to either the native or chloroplast-like Cys Northern blot analysis was carried out to test for the presence tRNA genes identified so far in plant mitochondria. The Cys in sugar beet mitochondria of RNAs corresponding to the trnC2-GCA is most likely the only functional tRNA gene in trnC1-GCA and trnC2-GCA sequences, respectively. tRNA- the sugar beet mitochondrial genome, although it remains to be Cys sized molecules (~80 bp) were detected by hybridization with seen whether the nuclear DNA-encoded tRNA species is a trnC2-GCA-specific probe, confirming transcription of this imported from the cytoplasm into mitochondria in sugar beet. 2576 Nucleic Acids Research, 2000, Vol. 28, No. 13 We have recently cloned a sugar beet cDNA encoding REFERENCES cysteinyl-tRNA synthase. 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Nucleic Acids Research – Oxford University Press

Published: Jul 1, 2000

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