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Plant Molecular Biology

, Volume 63, Issue 4, pp 519–532 | Cite as

Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.)

  • Dong Hwan Kim
  • Jeong Gu Kang
  • Byung-Dong KimEmail author
Article

Abstract

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T1 transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T1 plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper.

Keywords

Arabidopsis thaliana Capsicum annuum L. Cytoplasmic male sterility (CMS) Mitochondrial mutant Mitochondrial targeting signal orf456 

Notes

Acknowledgements

This research was supported by a grant from the Center for Plant Molecular Genetics and Breeding Research (CPMGBR) via the Korea Science and Engineering Foundation (KOSEF) and the Korea Ministry of Science and Technology (MOST). Dr. Yoo Jae Hyoung is acknowledged for the generous gift of plant material and discussions.

References

  1. Abad AR, Mehrtens BJ, Mackenzie SA (1995) Specific expression in reproductive tissues and fate of a mitochondrial sterility-associated protein in cytoplasmic male sterile bean. Plant Cell 7:271–285PubMedCrossRefGoogle Scholar
  2. Akagi H (1995) Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice. Theor Appl Genet 90:948–951CrossRefGoogle Scholar
  3. Akagi H, Sakamoto M, Shinjyo C, Shimada H, Fujimura T (1994) A unique sequence located downstream from the rice mitochondrial atp6 may cause male sterility. Curr Genet 25:52–58PubMedCrossRefGoogle Scholar
  4. Bonhomme S (1992) Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids. Mol Gen Genet 235:340–348PubMedCrossRefGoogle Scholar
  5. Bowman JL (1993) Arabidopsis: an atlas of morphology and development. Springer, Berlin Heidelberg New York, pp 277–283Google Scholar
  6. Chaumont F, Bernier B, Buxant R, Williams ME, Levings CS III, Boutry M (1995) Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration. Proc Natl Acad Sci USA 92:1167–1171PubMedCrossRefGoogle Scholar
  7. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  8. Cooper P, Butler E, Newton KJ (1990) Identification of a maize nuclear gene which influences the size and number of cox2 transcripts in mitochondria of perennial teosintes. Genetics 126:461–467,PubMedGoogle Scholar
  9. Dewey RE, Levings CS III, Timothy DH (1986) Novel recombinations in the maize mitochondrial genome produce a unique transcriptional unit in the Texas male-sterile cytoplasm. Cell 44:439–449PubMedCrossRefGoogle Scholar
  10. Dohmen G, Hessberg H, Geiger HH, Tudzynski P (1994) CMS in rye: comparative RFLP and transcript analyses of mitochondria from fertile and male-sterile plants. Theor Appl Genet 89:1014–1018CrossRefGoogle Scholar
  11. Ducos E, Touzet P, Boutry M (2001) The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes. Plant J 26:171–180PubMedCrossRefGoogle Scholar
  12. Grelon M, Budar F, Bonhomme S, Pelletier G (1994) Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids. Mol Gen Genet 243:540–547PubMedCrossRefGoogle Scholar
  13. Hanson MR (1991) Plant mitochondrial mutations and male sterility. Annu Rev Genet 25:461–486PubMedCrossRefGoogle Scholar
  14. Hanson M, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16:S154-S169PubMedCrossRefGoogle Scholar
  15. He S, Abad AR, Gelvin SB, Mackenzie SA (1996) A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco. Proc Natl Acad Sci USA 93:11763–11768PubMedCrossRefGoogle Scholar
  16. Iwabuchi M, Kyozuka J, Shimamoto K (1993) Processing followed by complete editing of an altered mitochondrial atp6 RNA restores fertility of cytoplasmic male sterile rice. EMBO J 12:1437–1446PubMedGoogle Scholar
  17. Johns C, Lu M, Lyznik A, Mackenzie S (1992) A mitochondrial DNA sequence is associated with abnormal pollen development in cytoplasmic male sterile bean plants. Plant Cell 4:435–449PubMedCrossRefGoogle Scholar
  18. Kanzaki H, Takeda M, Kameya T (1991) Sequence analysis of a mitochondrial DNA fragment isolated from cultured cells of carrot cytoplasmic male-sterile strain. Jpn J Genet 66:719–724PubMedCrossRefGoogle Scholar
  19. Kim DH, Kim BD (2005) Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.). Mol Cells 20:416–422PubMedGoogle Scholar
  20. Kim DH, Kim BD (2006a) The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.). Curr Genet 49:59–67CrossRefGoogle Scholar
  21. Kim DH, Kang JG, Kim SJ, Kim BD (2001) Identification of cox2 and atp6 regions as associated to CMS in Capsicum annuum by using RFLP and long accurate PCR. J Kor Soc Hortic Sci 42:121–127Google Scholar
  22. Kim DS, Kim DH, Yoo JH, Kim BD (2006) Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L). Mol Cells 21:135–140PubMedCrossRefGoogle Scholar
  23. Köhler RH, Horn R, Loss A, Zetsche K (1991) Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene. Mol Gen Genet 227:369–376PubMedCrossRefGoogle Scholar
  24. Köhler RH, Zipfel WR, Webb WW, Hanson MR (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11:613–621PubMedCrossRefGoogle Scholar
  25. Kubo T, Nishizawa S, Sugawara, Itchoda N, Estiati A, Mikami T (2000) The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNACys(GCA). Nucleic Acids Res 28:2571–2576PubMedCrossRefGoogle Scholar
  26. Kuhn J, Tengler U, Binder S (2001) Transcript lifetime is balanced between stabilizing stem loop structures and degradation promoting polyadenylation in plant mitochondria. Mol Cell Biol 21:731–742PubMedCrossRefGoogle Scholar
  27. Kuhn J, Binder S (2002) RT-PCR analysis of 5′ to 3′ end identifies mRNAs identifies the extremities of cox2 transcripts in pea mitochondria. Nucleic Acids Res 30:439–446PubMedCrossRefGoogle Scholar
  28. Lee JM, Nahm SH, Kim YM, Kim BD (2004) Characterization and molecular genetic mapping of microsatellite loci in pepper. Theor Appl Genet 108:619–627 PubMedCrossRefGoogle Scholar
  29. Levings III CS (1990) The Texas cytoplasm of maize: cytoplasmic male sterility and disease susceptibility. Science 250:942–947CrossRefPubMedGoogle Scholar
  30. Luo XD, Dai LF, Wang SB, Wolukau JN, Jahn M, Chen JF (2006) Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses. Plant Breed 125:395–399CrossRefGoogle Scholar
  31. Makaroff CA, Apel IJ, Palmer JD (1990) Characterization of radish mitochondrial atpA-associated sequences and relationship with male sterility. Plant Mol Biol 15:735–746PubMedCrossRefGoogle Scholar
  32. Menassa R, L’Homme Y, Brown GG (1999) Post-transcriptional and developmental regulation of a CMS-associated mitochondrial gene region by a nuclear restorer gene. Plant J 17:491–499PubMedCrossRefGoogle Scholar
  33. Millar AH, Sweetlove LJ, Giege P, Leaver CJ (2001) Analysis of the Arabidopsis Mitochondrial Proteome. Plant Physiol 127:1711–1727PubMedCrossRefGoogle Scholar
  34. Mohr S, Schulte-Kappert E, Odenbach W, Oettler G, Kück U (1993) Mitochondrial DNA of cytoplasmic male-sterile Triticum timopheevi: rearrangement of upstream sequences of the atp6 and orf25 genes. Theor Appl Genet 86:259–268CrossRefGoogle Scholar
  35. Moneger R, Smart CJ, Leaver CJ (1994) Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene. EMBO J 13:8–17PubMedGoogle Scholar
  36. Notsu Y, Masood S, Nishikawa T, Kubo N, Akiduki G, Nakazono M, Hirai A, Kadowaki K (2002) The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants. Mol Genet Genomics 268:434–445Google Scholar
  37. Novak F, Betiacb J, Dubovsky J (1971) Cytoplasmic male sterility in sweet pepper (Capsicum annuum L.). Biol Plant 12:275–280CrossRefGoogle Scholar
  38. Peterson PA (1958) Cytoplasmically inherited male sterility in Capsicum. Am Nat 92:111–119CrossRefGoogle Scholar
  39. Piffanelli P, Ross HE, Murphy DJ (1998) Biogenesis and function of the lipidic structures of pollen grains. Sex Plant Reprod 11:65–80CrossRefGoogle Scholar
  40. Prutt KD, Hanson MR (1989) Cytochrome oxydase subunit II sequences in petunia mitochondria: two intron-containing genes and an intron-less pseudogene associated with cytoplasmic male sterility. Curr Genet 16:281–291CrossRefGoogle Scholar
  41. Sane AP, Nath P, Sane PV (1994) Mitochondrial ATP synthase genes may be implicated in cytoplasmic male sterility in sorghum bicolor. J Biosci 19:43–55Google Scholar
  42. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  43. Schnable PS, Wise RP (1998) The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci 3:175–180CrossRefGoogle Scholar
  44. Singh M, Hamel N, Menassa R, Li XQ, Young B, Jean M, Landry BS, Brown GG (1996) Nuclear genes associated with a single Brassica CMS restorer locus influence transcripts of three different mitochondrial gene regions. Genetics 143:505–516PubMedGoogle Scholar
  45. Spassova M, Moneger F, Leaver CJ, Petrov P, Atanassov A, Nijkamp HJ, Hille J (1994) Characterization and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower. Plant Mol Biol 26:1819–1831PubMedCrossRefGoogle Scholar
  46. Sugiyama Y, Watase Y, Nagase M, Makita N, Yagura S, Hirai A, Sugiura M (2005) The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants. Mol Gen Genomics 272:603–615CrossRefGoogle Scholar
  47. Tang HV, Pring DR, Shaw LC, Salazar RA, Muza FR, Yan B, Schertz KF (1996) Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile Sorghum. Plant J 10:123–133PubMedCrossRefGoogle Scholar
  48. Unseld M, Marienfeld JR, Brandt P, Brennicke A (1997) The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nat Genet 15:57–61PubMedCrossRefGoogle Scholar
  49. Wang LH, Zhang BX, Lefebvre V, Huang SW, Daubèze AM, Palloix A (2004) QTL analysis of fertility restoration in cytoplasmic male sterile pepper. Theor Appl Genet 109:1058–1063PubMedCrossRefGoogle Scholar
  50. Wintz H, Chen HC, Sutton CA, Conley CA, Cobb A, Ruth D, Hanson MR (1995) Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco. Plant Mol Biol 28:83–92PubMedCrossRefGoogle Scholar
  51. Young EG, Hanson MR (1987) A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated. Cell 50:41–49PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Dong Hwan Kim
    • 1
  • Jeong Gu Kang
    • 1
  • Byung-Dong Kim
    • 1
    Email author
  1. 1.Department of Plant Science, College of Agriculture and Life Sciences, and Center for Plant Molecular Genetics and Breeding ResearchSeoul National UniversitySeoulKorea

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