Current Genetics

, Volume 62, Issue 2, pp 431–442 | Cite as

The linear plastid chromosomes of maize: terminal sequences, structures, and implications for DNA replication

  • Delene J. Oldenburg
  • Arnold J. BendichEmail author
Original Article


The structure of a chromosomal DNA molecule may influence the way in which it is replicated and inherited. For decades plastid DNA (ptDNA) was believed to be circular, with breakage invoked to explain linear forms found upon extraction from the cell. Recent evidence indicates that ptDNA in vivo consists of linear molecules with discrete termini, although these ends were not characterized. We report the sequences of two terminal regions, End1 and End2, for maize (Zea mays L.) ptDNA. We describe structural features of these terminal regions and similarities found in other plant ptDNAs. The terminal sequences are within inverted repeat regions (leading to four genomic isomers) and adjacent to origins of replication. Conceptually, stem-loop structures may be formed following melting of the double-stranded DNA ends. Exonuclease digestion indicates that the ends in maize are unobstructed, but tobacco (Nicotiana tabacum L.) ends may have a 5′-protein. If the terminal structure of ptDNA molecules influences the retention of ptDNA, the unprotected molecular ends in mature leaves of maize may be more susceptible to degradation in vivo than the protected ends in tobacco. The terminal sequences and cumulative GC skew profiles are nearly identical for maize, wheat (Triticum aestivum L.) and rice (Oryza sativa L.), with less similarity among other plants. The linear structure is now confirmed for maize ptDNA and inferred for other plants and suggests a virus-like recombination-dependent replication mechanism for ptDNA. Plastid transformation vectors containing the terminal sequences may increase the chances of success in generating transplastomic cereals.


GC skew Herpes simplex virus Chloroplast DNA Recombination-dependent replication Telomeres 



Dilution buffer


High salt buffer


Herpes simplex virus


Inverted repeat


Long single copy


Mitochondrial DNA


Origin binding protein


Origin of replication


Pulsed-field gel electrophoresis


Proteinase K


Plastid DNA


Recombination-dependent replication


Single-strand binding protein


Short single copy


Single-strand annealing



This research was funded by the Junat Fund (a private charitable fund).

Compliance with ethical standards

Conflict of interest

The authors DO and AB are co-inventers for the patent application: “Plastid Transformation Using Linear DNA Vectors”; application nos. PCT/US2013/030775, WO2014/065857A1. The linear vectors described in this patent are based on the terminal ptDNA sequences reported in this manuscript.

Supplementary material

294_2015_548_MOESM1_ESM.pdf (1.3 mb)
Supplementary material 1 (PDF 1370 kb) Online Resource 1: Comparisons of maize ptDNA end sequences to sequences from other ptDNAs; sequence alignments and amount of similarity are shown
294_2015_548_MOESM2_ESM.pdf (366 kb)
Supplementary material 2 (PDF 365 kb) Online Resource 2: GC skew plots of the plastid genomes from several land plants and the protist Euglena gracilis and viral genome of herpes simplex
294_2015_548_MOESM3_ESM.pdf (855 kb)
Supplementary material 3 (PDF 855 kb) Online Resource 3: Exonuclease digestion and PFGE of maize and tobacco ptDNAs
294_2015_548_MOESM4_ESM.pdf (382 kb)
Supplementary material 4 (PDF 383 kb) Online Resource 4: Secondary structure prediction of the terminal sequences from maize, rice, and wheat


  1. Aslani A, Simonsson S, Elias P (2000) A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein. J Biol Chem 275:5880–5887CrossRefPubMedGoogle Scholar
  2. Belanger AS, Brouard JS, Charlebois P, Otis C, Lemieux C, Turmel M (2006) Distinctive architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium helveticum. Mol Genet Genomics 276:464–477CrossRefPubMedGoogle Scholar
  3. Bendich AJ (1996) Structural analysis of mitochondrial DNA molecules from fungi and plants using moving pictures and pulsed-field gel electrophoresis. J Mol Biol 255:564–588CrossRefPubMedGoogle Scholar
  4. Bendich AJ (2004) Circular chloroplast chromosomes: the grand illusion. Plant Cell 16:1661–1666CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bendich AJ (2007) The size and form of chromosomes are constant in the nucleus, but highly variable in bacteria, mitochondria and chloroplasts. Bioessays 29:474–483CrossRefPubMedGoogle Scholar
  6. Bendich AJ, Oldenburg DJ (2013) Plastid transformation using linear DNA vectors. I. B. World Intellectual Property Organization, USA, pp 1–59Google Scholar
  7. Bendich AJ, Smith SB (1990) Moving pictures and pulsed-field gel electrophoresis show linear DNA molecules from chloroplasts and mitochondria. Curr Genet 17:421–425CrossRefGoogle Scholar
  8. Bochman ML, Paeschke K, Zakian VA (2012) DNA secondary structures: stability and function of G-quadruplex structures. Nat Rev Genet 13:770–780CrossRefPubMedPubMedCentralGoogle Scholar
  9. Boulikas T (1996) Common structural features of replication origins in all life forms. J Cell Biochem 60:297–316CrossRefPubMedGoogle Scholar
  10. Brouard JS, Otis C, Lemieux C, Turmel M (2011) The chloroplast genome of the green alga Schizomeris leibleinii (Chlorophyceae) provides evidence for bidirectional DNA replication from a single origin in the chaetophorales. Genome Biol Evol 3:505–515CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chaconas G, Kobryn K (2010) Structure, function, and evolution of linear replicons in Borrelia. Annu Rev Microbiol 64:185–202CrossRefPubMedGoogle Scholar
  12. Clarke JL, Daniell H, Nugent JM (2011) Chloroplast biotechnology, genomics and evolution: current status, challenges and future directions. Plant Mol Biol 76:207–209CrossRefPubMedPubMedCentralGoogle Scholar
  13. Deng X-W, Wing RA, Gruissem W (1989) The chloroplast genome exists in multimeric forms. Proc Natl Acad Sci U S A 86:4156–4160CrossRefPubMedPubMedCentralGoogle Scholar
  14. Deng SK, Chen H, Symington LS (2015) Replication protein A prevents promiscuous annealing between short sequence homologies: implications for genome integrity. Bioessays 37:305–313CrossRefPubMedGoogle Scholar
  15. Gerhold JM, Sedman T, Visacka K, Slezakova J, Tomaska L, Nosek J, Sedman J (2014) Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria. J Biol Chem 289:22659–22670CrossRefPubMedPubMedCentralGoogle Scholar
  16. Grigoriev A (1998) Analyzing genomes with cumulative skew diagrams. Nucleic Acids Res 26:2286–2290CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hanson MR, Gray BN, Ahner BA (2013) Chloroplast transformation for engineering of photosynthesis. J Exp Bot 64:731–742CrossRefPubMedGoogle Scholar
  18. Heinhorst S, Cannon GC (1993) DNA replication in chloroplasts. J Cell Sci 104:1–9Google Scholar
  19. Kikin O, D’Antonio L, Bagga PS (2006) QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucl Acids Res 34(suppl 2):W676–W682CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kolodner RD, Tewari KK (1975) Chloroplast DNA from higher plants replicates by both the Cairns and the rolling circle mechanism. Nature 256:708–711CrossRefPubMedGoogle Scholar
  21. Kunnimalaiyaan M, Nielsen BL (1997) Chloroplast DNA replication: mechanism, enzymes and replication origins. J Plant Biochem Biotechnol 6:1–7CrossRefGoogle Scholar
  22. Lassen MG, Kochhar S, Nielsen BL (2011) Identification of a soybean chloroplast DNA replication origin-binding protein. Plant Mol Biol 76:463–471CrossRefPubMedGoogle Scholar
  23. Lilly JW, Havey MJ, Jackson SA, Jiang J (2001) Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants. Plant Cell 13:245–254CrossRefPubMedPubMedCentralGoogle Scholar
  24. Maier RM, Neckerman K, Igloi GL, Kössel H (1995) Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. J Mol Biol 251:614–628CrossRefPubMedGoogle Scholar
  25. Majeran W, Friso G, Asakura Y, Qu X, Huang M, Ponnala L, Watkins KP, Barkan A, van Wijk KJ (2012) Nucleoid-enriched proteomes in developing plastids and chloroplasts from maize leaves: a new conceptual framework for nucleoid functions. Plant Physiol 158:156–189CrossRefPubMedGoogle Scholar
  26. Maliga P, Bock R (2011) Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol 155:1501–1510CrossRefPubMedPubMedCentralGoogle Scholar
  27. Marechal A, Brisson N (2010) Recombination and the maintenance of plant organelle genome stability. New Phytol 186:299–317CrossRefPubMedGoogle Scholar
  28. Moriyama T, Sato N (2014) Enzymes involved in organellar DNA replication in photosynthetic eukaryotes. Front Plant Sci 5:480CrossRefPubMedPubMedCentralGoogle Scholar
  29. Morton BR (1999) Strand asymmetry and codon usage bias in the chloroplast genome of Euglena gracilis. Proc Natl Acad Sci U S A 96:5123–5128CrossRefPubMedPubMedCentralGoogle Scholar
  30. Muylaert I, Tang KW, Elias P (2011) Replication and recombination of herpes simplex virus DNA. J Biol Chem 286:15619–15624CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nosek J, Kosa P, Tomaska L (2006) On the origin of telomeres: a glimpse at the pre-telomerase world. Bioessays 28:182–190CrossRefPubMedGoogle Scholar
  32. Oldenburg DJ, Bendich AJ (2001) Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5′ protein. J Mol Biol 310:549–562CrossRefPubMedGoogle Scholar
  33. Oldenburg DJ, Bendich AJ (2004a) Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms. J Mol Biol 335:953–970CrossRefPubMedGoogle Scholar
  34. Oldenburg DJ, Bendich AJ (2004b) Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize. J Mol Biol 344:1311–1330CrossRefPubMedGoogle Scholar
  35. Oldenburg DJ, Bendich AJ (2009) Chloroplasts. In: Kriz AL, Larkins BA (eds) Molecular Genetic Approaches to Maize Improvement Biotechnology in agriculture and forestry, vol 63. Springer, Berlin, Heidelberg, pp 325–343Google Scholar
  36. Oldenburg DJ, Bendich AJ (2015) DNA maintenance in plastids and mitochondria of plants. Front Plant Sci 6:883. doi: 10.3389/fpls.2015.00883 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Oldenburg DJ, Rowan BA, Zhao L, Walcher CL, Schleh M, Bendich AJ (2006) Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype. Planta 225:41–55CrossRefPubMedGoogle Scholar
  38. Oldenburg DJ, Rowan BA, Kumar RA, Bendich AJ (2014) On the fate of plastid DNA molecules during leaf development: response to the Golczyk et al. Commentary. Plant Cell 26:855–861CrossRefPubMedPubMedCentralGoogle Scholar
  39. Pearson CE, Zorbas H, Price GB, Zannis-Hadjopoulos M (1996) Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication. J Cell Biochem 63:1–22CrossRefPubMedGoogle Scholar
  40. Pohjoismaki JL, Goffart S (2011) Of circles, forks and humanity: topological organisation and replication of mammalian mitochondrial DNA. Bioessays 33:290–299CrossRefPubMedGoogle Scholar
  41. Ravi V, Khuranan JP, Tyagi AK, Khurnana P (2008) An update on chloroplast genomes. Plant Syst Evol 271:101–122CrossRefGoogle Scholar
  42. Rowan BA, Oldenburg DJ, Bendich AJ (2004) The demise of chloroplast DNA in Arabidopsis. Curr Genet 46:176–181CrossRefPubMedGoogle Scholar
  43. Rowan BA, Oldenburg DJ, Bendich AJ (2010) RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis. J Exp Bot 61:2575–2588CrossRefPubMedPubMedCentralGoogle Scholar
  44. Sakaguchi K, Ishibashi T, Uchiyama Y, Iwabata K (2009) The multi-replication protein A (RPA) system—a new perspective. FEBS J 276:943–963CrossRefPubMedGoogle Scholar
  45. Scharff LB, Koop HU (2006) Linear molecules of tobacco ptDNA end at known replication origins and additional loci. Plant Mol Biol 62:611–621CrossRefPubMedGoogle Scholar
  46. Scharff LB, Koop HU (2007) Targeted inactivation of the tobacco plastome origins of replication A and B. Plant J 50:782–794CrossRefPubMedGoogle Scholar
  47. Sharma S (2011) Non-B DNA secondary structures and their resolution by RecQ helicases. J Nucleic Acids 2011:724215CrossRefPubMedPubMedCentralGoogle Scholar
  48. Shaver JM, Oldenburg DJ, Bendich AJ (2006) Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize. Planta 224:72–82CrossRefPubMedGoogle Scholar
  49. Shaver JM, Oldenburg DJ, Bendich AJ (2008) The structure of chloroplast DNA molecules and the effects of light on the amount of chloroplast DNA during development in Medicago truncatula. Plant Physiol 146:1064–1074CrossRefPubMedPubMedCentralGoogle Scholar
  50. Smith DR, Keeling PJ (2013) Gene conversion shapes linear mitochondrial genome architecture. Genome Biol Evol 5:905–912CrossRefPubMedPubMedCentralGoogle Scholar
  51. Suzuki JY, Sriraman P, Svab Z, Maliga P (2003) Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants. Plant Cell 15:195–205CrossRefPubMedPubMedCentralGoogle Scholar
  52. Watson JD (1972) Origin of concatemeric T7 DNA. Nat New Biol 239:197–201CrossRefPubMedGoogle Scholar
  53. Weller SK, Coen DM (2012) Herpes simplex viruses: mechanisms of DNA replication. Cold Spring Harb Perspect Biol 4:a013011CrossRefPubMedPubMedCentralGoogle Scholar
  54. Weller SK, Sawitzke JA (2014) Recombination promoted by DNA viruses: phage lambda to herpes simplex virus. Annu Rev Microbiol 68:237–258CrossRefPubMedPubMedCentralGoogle Scholar
  55. Xia X (2012) DNA replication and strand asymmetry in prokaryotic and mitochondrial genomes. Curr Genomics 13:16–27CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of BiologyUniversity of WashingtonSeattleUSA

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