Molecular Genetics and Genomics

, Volume 290, Issue 1, pp 1–9 | Cite as

The application of RNA-seq to the comprehensive analysis of plant mitochondrial transcriptomes

  • James D. Stone
  • Helena StorchovaEmail author


We review current studies of plant mitochondrial transcriptomes performed by RNA-seq, highlighting methodological challenges unique to plant mitochondria. We propose ways to improve read mapping accuracy and sensitivity such as modifying a reference genome at RNA editing sites, using splicing- and ambiguity-competent aligners, and masking chloroplast- or nucleus-derived sequences. We also outline modified RNA-seq methods permitting more accurate detection and quantification of partially edited sites and the identification of transcription start sites on a genome-wide scale. The application of RNA-seq goes beyond genome-wide determination of transcript levels and RNA maturation events, and emerges as an elegant resource for the comprehensive identification of editing, splicing, and transcription start sites. Thus, improved RNA-seq methods customized for plant mitochondria hold tremendous potential for advancing our understanding of plant mitochondrial evolution and cyto-nuclear interactions in a broad array of plant species.


RNA-seq Plant mitochondria Transcriptome Editing 



We are grateful to Daniel B Sloan for reading and commenting on this manuscript. This work originated in the framework of the project “Integration of the experimental and population biology using new methods of interdisciplinary issues-the way to excellence with young scientists,” Reg.No.: CZ.1.07/2.3.00/30.0048, funded by the European Social Fund (ESF) and the state budget of Czech Republic through the Operational Programme Education for Competitiveness (OPEC). It was further supported by the grant of the Grant Agency of the Czech Republic P506/12/1359 to H.S.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Adamo A, Pinney JW, Kunova A, Westhead DR, Meyer P (2008) Heat stress enhances the accumulation of polyadenylated mitochondrial transcripts in Arabidopsis thaliana. PLoS One 3:e2889. doi:  10.1371/journal.pone.0002889
  2. Anderson L (1981) Identification of mitochondrial proteins and some of their precursors in two-dimension a electrophoretic maps of human cells. Proc Natl Acad Sci USA 78:2407–2411. doi: 10.1073/pnas.78.4.2407 PubMedCentralPubMedCrossRefGoogle Scholar
  3. Bentolila S, Oh J, Hanson MR, Bukowski R (2013) Comprehensive high-resolution Analysis of the role of an Arabidopsis gene family in RNA editing. PLoS Genet 9:e1003584. doi: 10.1371/journal.pgen.1003584 PubMedCentralPubMedCrossRefGoogle Scholar
  4. Case AL, Willis JH (2008) Hybrid male sterility in Mimulus (Phrymaceae) is associated with a geographically restricted mitochondrial rearrangement. Evolution 62:1026–1039. doi: 10.1111/j.1558-5646.2008.00360.x PubMedCrossRefGoogle Scholar
  5. Chang JH, Tong L (2012) Mitochondrial poly(A) polymerase and polyadenylation. Biochim Biophys Acta 1819:992–997. doi: 10.1016/j.bbagrm.2011.10.012 PubMedCentralPubMedCrossRefGoogle Scholar
  6. Das S, Sen S, Chakraborty A, Chakraborti P, Maiti MK, Basu A, Basu D, Sen SK (2010) An unedited 1.1 kb mitochondrial orfB gene transcript in the Wild Abortive Cytoplasmic Male Sterility (WA-CMS) system of Oryza sativa L. subsp indica. BMC Plant Biol 10:39. doi: 10.1186/1471-2229-10-39 PubMedCentralPubMedCrossRefGoogle Scholar
  7. DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43:491–498. doi: 10.1038/ng.806 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21. doi: 10.1093/bioinformatics/bts635 PubMedCentralPubMedCrossRefGoogle Scholar
  9. Fang Y, Wu H, Zhang T, Yang M, Yin Y, Pan L, Yu X, Zhang X, Hu S, Al-Mssallem IS, Yu J (2012) A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome. PLoS One 7:e37164. doi: 10.1371/journal.pone.0037164 PubMedCentralPubMedCrossRefGoogle Scholar
  10. Forner J, Weber B, Wietholter C, Meyer RC, Binder S (2005) Distant sequences determine 5′ end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24. Nucleic Acids Res 33:4673–4682. doi: 10.1093/nar/gki774 PubMedCentralPubMedCrossRefGoogle Scholar
  11. Forner J, Weber B, Thuss S, Wildum S, Binder S (2007) Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5′and 3′ end formation. Nucleic Acids Res 35:3676–3692. doi: 10.1093/nar/gkm270 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Fujii S, Toda T, Kikuchi S, Suzuki R, Yokoyama K, Tsuchida H, Yano K, Toriyama K (2011) Transcriptome map of plant mitochondria reveals islands of unexpected transcribed regions. BMC Genom 12:279. doi: 10.1186/1471-2164-12-279 CrossRefGoogle Scholar
  13. Gagliardi D, Leaver JCJ (1999) Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower. EMBO J 18:3757–3766. doi: 10.1093/emboj/18.13.3757 PubMedCentralPubMedCrossRefGoogle Scholar
  14. German MA, Pillay M, Jeong DH, Hetawal A, Luo SJ, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R et al (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNAends. Nat Biotechnol 26:941–946. doi: 10.1038/nbt1417 PubMedCrossRefGoogle Scholar
  15. Gott JM, Emeson RB (2000) Functions and mechanisms of RNA editing. Annu Rev Genet 34: 499–U34. doi: 10.1146/annurev.genet.34.1.499
  16. Grewe F, Herres S, Viehöver P, Polsakiewicz M, Weisshaar B, Knoop V (2011) A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii. Nucl Acids Res 39:2890–2902. doi: 10.1093/nar/gkq1227 PubMedCentralPubMedGoogle Scholar
  17. Grewe F, Edger PP, keren I, Sultan L, Pires JC, Ostersetzer-Biran O, Mower JP (2014) Comparative analysis of 11 Brassicales mitochondrial genomes and the mitochondrial transcriptome of Brassica oleracea. Mitochondrion. doi: 10.1016/j.mito.2014.05.008 PubMedGoogle Scholar
  18. Grimes BT, Sisay AK, Carroll HD, Cahoon AB (2014) Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding region. BMC Genom 15:31. doi: 10.1186/1471-2164-15-31 CrossRefGoogle Scholar
  19. Handa H, Gualberto JM, Grienenberg JM (1995) Characterization of the mitochondrial orfB gene and its derivative, orf224, a chimeric open reading frame specific to one mitochondrial genome of the Polima male-sterile cytoplasm in rapeseed (Brassica napus L.). Curr Genet 28:546–552. doi: 10.1007/BF00518167 PubMedCrossRefGoogle Scholar
  20. Hirayama T, Matsuura T, Ushiyama S, Narusaka M, Kurihara Y, Yasuda M, Ohtani M, seki M, demura T, Nakashita H, Narusaka Y, Hayashi S (2013) A poly(A)-specific ribonuclease directly regulates the poly(A) status of mitochondrial mRNA in Arabidopsis. Nat Commun 4:2247. doi: 10.1038/ncomms3247 PubMedGoogle Scholar
  21. Howad W, Kempken F (1997) Cell type-specific loss of at p6 RNA editing in cytoplasmic male sterile Sorghum bicolor. Proc Natl Acad Sci USA 94:11090–11095. doi: 10.1073/pnas.94.20.11090 PubMedCentralPubMedCrossRefGoogle Scholar
  22. Hu JH, Yi R, Zhang HY, Ding Y (2013) Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines. Mitochondrion 13:87–95. doi: 10.1016/j.mito.2013.01.011 PubMedCrossRefGoogle Scholar
  23. Hu J, Huang W, Huang Q, Qin X, Yu C, Wang L, Li S, Zhu R, Zhu Y (2014) Mitochondria and cytoplasmic male sterility in plants. Mitochondrion. doi: 10.1016/j.mito.2014.02.008
  24. Islam MS, Studer B, Byrne SL, Farrell JD, Panitz F, Bendixen C, Moller IA, Asp T (2013) The genome and transcriptome of perennial ryegrass mitochondria. BMC Genom 14:202. doi: 10.1186/1471-2164-14-202 CrossRefGoogle Scholar
  25. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36. doi: 10.1186/gb-2013-14-4-r36 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Knoop V (2011) When you can’t trust the DNA: rNA editing changes transcript sequences. Cell Mol Life Sci 68:567–586. doi: 10.1007/s00018-010-0538-9 PubMedCrossRefGoogle Scholar
  27. Kubo T, Nishizawa S, Mikami T (1999) Alterations in organization and transcription of the mitochondrial genome of cytoplasmic male sterile sugar beet (Beta vulgaris L.). Mol Gen Genet 262:283–290. doi: 10.1007/s004380051085 PubMedCrossRefGoogle Scholar
  28. Kühn J, Binder S (2002) RT-PCR analysis of 5′ to 3′ end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria. Nucleic Acids Res 30:439–446. doi: 10.1093/nar/30.2.439 PubMedCentralPubMedCrossRefGoogle Scholar
  29. Kühn K, Weihe A, Börner T (2005) Multiple promoters are a common feature of mitochondrial genes in Arabidopsis. Nucleic Acids Res 33:337–346. doi: 10.1093/nar/gki179 PubMedCentralPubMedCrossRefGoogle Scholar
  30. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923 PubMedCentralPubMedCrossRefGoogle Scholar
  31. Lee J-H, Ang JK, Xiao X (2013) Analysis and design of RNA sequencing experiments for identifying RNA editing and other single-nucleotide variants. RNA 19:725–732. doi: 10.1261/rna.037903.112 PubMedCentralPubMedCrossRefGoogle Scholar
  32. Lee W-P, Stromberg MP, Ward A, Steward C, Garrison EP, Marth GT (2014) MOSAIK: a Hash-Based Algorithm for Accurate Next-Generation Sequencing Short-Read Mapping. PLoS ONE 9(3):e90581. doi: 10.1371/journal.pone.0090581 PubMedCentralPubMedCrossRefGoogle Scholar
  33. Leino M, Landgren M, Glimelius K (2005) Alloplasmic effects on mitochondrial transcriptional activity and RNA turnover result in accumulated transcripts of Arabidopsis orfs in cytoplasmic male-sterile Brassica napus. Plant J 42:469–480. doi: 10.1111/j.1365-313X.2005.02389.x PubMedCrossRefGoogle Scholar
  34. Lim J-Q, Tennakoon C, Li G, Wong E, Ruan Y, Wei C-L, Sung W-K (2012) BatMeth: improved mapper for bisulfite sequencing reads on DNA methylation. Genome Biol 13:R82. doi: 10.1186/gb-2012-13-10-r82 PubMedCentralPubMedCrossRefGoogle Scholar
  35. Mercer TM, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood AMJ, Haugen E, Bracken CP, Rackham O, Stamatoyannopoulos JA, Filipovska A, Mattick JS (2011) The human mitochondrial transcriptome. Cell 146:645–658. doi: 10.1016/j.cell2011.06.051 PubMedCentralPubMedCrossRefGoogle Scholar
  36. Michalovova M, Vyskot B, Kejnovsky E (2013) Analysis of plastid and mitochondrial DNA insertions in the nucleus (NUPTs and NUMTs) of six plant species: size, relative age and chromosomal localization. Heredity 111:314–320. doi: 10.1038/hdy.2013.51 PubMedCentralPubMedCrossRefGoogle Scholar
  37. Mower JP (2008) Modeling sites of RNA editing as a fifth nucleotide state reveals progressive loss of edited sites from angiosperm mitochondria. Mol Biol Evol 25:52–61. doi: 10.1093/molbev/msm226 PubMedCrossRefGoogle Scholar
  38. Mower JP (2009) The PREP suite: predictive RNA editors for plant mitochondrial genes, chloroplast genes and user-defined alignments. Nucleic Acids Res 37:W253–W259. doi: 10.1093/nar/gkp337 PubMedCentralPubMedCrossRefGoogle Scholar
  39. Müller K, Storchova H (2013) Transcription of atp1 is influenced by both genomic configuration and nuclear background in the highly rearranged mitochondrial genomes of Silene vulgaris. Plant Mol Biol 81:495–505. doi: 10.1007/s11103-013-0018-3 PubMedCrossRefGoogle Scholar
  40. Okazaki M, Kazama T, Murata H, Motomura K, Toriyama K (2013) Whole mitochondrial genome sequencing and transcriptional analysis to uncover an rt102-type cytoplasmic male sterility-associated candidate gene derived from Oryza rufipogon. Plant Cell Physiol 54:1560–1568. doi: 10.1093/pcp/pct102 PubMedCrossRefGoogle Scholar
  41. Picardi E, Horner DS, Chiara M, Schiavon R, Valle G, Pesole G (2010) Large-scale detection and analysis of RNA editing in grape mtDNA by RNA deep-sequencing. Nucleic Acids Res 38:4755–4767. doi: 10.1093/nar/gkq202 PubMedCentralPubMedCrossRefGoogle Scholar
  42. Preuten T, Cincu E, Fuchs J, Zoschke R, Liere K, Boerner T (2010) Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells. Plant J64:948–959. doi: 10.1111/j.1365-313X.2010.04389.x CrossRefGoogle Scholar
  43. Richardson AO, Rice DW, Young GJ, Alverson AJ, Palmer JD (2013) The “fossilized” mitochondrial genome of Liriodendron tulipifera: ancestral gene content and order, ancestral editing sites, and extraordinarily low mutation rate. BMC Biol 11:29. doi: 10.1186/1741-7007-11-29 PubMedCentralPubMedCrossRefGoogle Scholar
  44. Ruwe H, Schmitz-Linneweber C (2012) Short non-coding RNA fragments accumulating in chloroplasts: footprints of RNA binding proteins? Nucleic Acids Res 40:3106–3116. doi: 10.1093/nar/gkr1138 PubMedCentralPubMedCrossRefGoogle Scholar
  45. Ruwe H, Castandet B, Schmitz-Linneweber C, Stern DB (2013) Arabidopsis chloroplast quantitative editotype. FEBS Lett 587:1429–1433. doi: 10.1016/j.febslet.2013.03.022 PubMedCrossRefGoogle Scholar
  46. Salmans ML, Chaw SM, Lin CP, Shih ACC, Wu YW, Mulligan RM (2010) Editing site analysis in a gymnosperm mitochondrial genome reveals similarities with angiosperm mitochondrial genomes. Curr Genet 56:439–446. doi: 10.1007/s00294-010-0312-4 PubMedCentralPubMedCrossRefGoogle Scholar
  47. Satya RV, Zavaljevski N, Reifman J (2012) A new strategy to reduce allelic bias in RNA-Seq readmapping. Nucleic Acids Res 40:e127. doi: 10.1093/nar/gks425 PubMedCrossRefGoogle Scholar
  48. Shearman JR, Sangsrakru D, Ruang-Areerate P, Sonthirod C, Uthaipaisanwong P, Yoocha T, Poopear S, Theerawattanasuk K, Tragoonrung S, Tangphatsornruang S (2014) Assembly and analysis of a male sterile rubber tree mitochondrial genome reveals DNA rearrangement events and a novel transcript. BMC Plant Biol 14:45. doi: 10.1186/1471-2229-14-45 PubMedCentralPubMedCrossRefGoogle Scholar
  49. Sloan DB (2013) One ring to rule them all? Genome sequencing provides new insights into the ‘master circle’model of plant mitochondrial DNA structure. New Phytol 200:978–985. doi: 10.1111/nph.12395 PubMedCrossRefGoogle Scholar
  50. Sloan DB, Alverson AJ, Štorchová H, Palmer JD, Taylor DR (2010a) Extensive loss of translational genes in the structurally dynamic mitochondrial genome of the angiosperm Silene latifolia. BMC Evol Biol 10:274. doi: 10.1186/1471-2148-10-274 PubMedCentralPubMedCrossRefGoogle Scholar
  51. Sloan DB, MacQueen AH, Alverson AJ, almer JD, Taylor DR (2010b) Extensive loss of rna editing sites in rapidly evolving silene mitochondrial genomes: selection vs. retroprocessing as the driving force. Genetics 185: 1369-U358. doi: 10.1534/genetics.110.118000
  52. Sloan DB, Alverson AJ, Chuckalovcak JP, Wu M, McCauley DE, Palmer JD, Taylor DR (2012) Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates. PLoS Biol 10:e1001241. doi: 10.1371/journal.pbio.1001241 PubMedCentralPubMedCrossRefGoogle Scholar
  53. Stevenson KR, Coolon JD, Wittkopp PJ (2013) Sources of bias in measures of allele-specific expression derived from RNA-seq data aligned to a single reference genome. BMC Genom 14:536. doi: 10.1186/1471-2164-14-536 CrossRefGoogle Scholar
  54. Storchova H, Müller K, Lau S, Olson MS (2012) Mosaic origin of a complex chimeric mitochondrial gene in Silene vulgaris. PLoS One 7:e30401. doi:  10.1371/journal.pone.0030401
  55. Takenaka M, Verbitskiy D, Zehrmann A, Hartel B, Bayer-Csaszar E, Glass F, Brennicke A (2014) RNA editing in plant mitochondria—Connecting RNA target sequences and acting proteins. Mitochondrion. doi: 10.1016/j.mito.2014.04.005 Google Scholar
  56. Terachi T, Yamaguchi K, Yamagishi H (2001) Sequence analysis on the mitochondrial orfB locus in normal and Ogura male-sterile cytoplasms from wild and cultivated radishes. Curr Genet 40:276–281. doi: 10.1007/s00294-001-0256-9 PubMedCrossRefGoogle Scholar
  57. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella KV, Altshuler D, Gabriel S, DePristo MA (2013) From fastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr Protoc Bioinform 43:11.10.1–11.10.33. doi: 10.1002/0471250953.bi1110s43
  58. Woloczynska M, Kmiec B, Mackiewicz P, Janska H (2006) Copy number of bean mitochondrial genes estimated by real-time PCR does not correlate with the number of gene loci and transcript levels. Plant Mol Biol 61:1–12. doi: 10.1007/s11103-005-5773-3 CrossRefGoogle Scholar
  59. Wu TD, Nacu S (2010) Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26:873–881. doi: 10.1093/bioinformatics/btq057 PubMedCentralPubMedCrossRefGoogle Scholar
  60. Zhang QY, Liu YG (2006) Rice mitochondrial genes are transcribed by multiple promoters that are highly diverged. J Integr Plant Biol 48:1473–1477. doi: 10.1111/j.1672-9072.2006.00384.x CrossRefGoogle Scholar
  61. Zhelyazkova P, Sharma CM, Fortsner KU, Liere K, Vogel J, Boerner T (2012) The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase. Plant Cell 24:123–136. doi: 10.1105/tpc.111.089441 PubMedCentralPubMedCrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Institute of Experimental Botany v.v.iAcademy of Sciences of the Czech RepublicPragueCzech Republic
  2. 2.Institute of Botany v.v.iAcademy of Sciences of the Czech RepublicPrague WestCzech Republic

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