Skip to main content
Log in

Ty3/gypsy-like retrotransposon knockout of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase is non-lethal, uncovers a cryptic paralogous mutation, and produces novel tocopherol (vitamin E) profiles in sunflower

  • Original Paper
  • Published:
Theoretical and Applied Genetics Aims and scope Submit manuscript

Abstract

The m (Tph 1) mutation partially disrupts the synthesis of α-tocopherol (vitamin E) in sunflower (Helianthus annuus L.) seeds and was predicted to disrupt a methyltransferase activity necessary for the synthesis of α- and γ-tocopherol. We identified and isolated two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs from sunflower (MT-1 and MT-2), resequenced MT-1 and MT-2 alleles from wildtype (m + m +) and mutant (m m) inbred lines, identified m as a non-lethal knockout mutation of MT-1 caused by the insertion of a 5.2 kb Ty3/gypsy-like retrotransposon in exon 1, and uncovered a cryptic codominant mutation (d) in a wildtype × mutant F2 population predicted to be segregating for the m mutation only. MT-1 and m cosegregated and mapped to linkage group 1 and MT-1 was not transcribed in mutant homozygotes (m m). The m locus was epistatic to the d locus—the d locus had no effect in m + m + and m + m individuals, but significantly increased β-tocopherol percentages in m m individuals. MT-2 and d cosegregated, MT-2 alleles isolated from mutant homozygotes (d d) carried a 30 bp insertion at the start of the 5′-UTR, and MT-2 was more strongly transcribed in seeds and leaves of wildtype (d + d +) than mutant (d d) homozygotes (transcripts were 2.2- to 5.0-fold more abundant in the former than the latter). The double mutant (m m d d) was non-lethal and produced 24–45% α- and 55–74% β-tocopherol (the wildtype produced 96% α- and 4% β-tocopherol). MT-2 compensated for the loss of the MT-1 function, and the MT-2 mutation profoundly affected the synthesis of tocopherols without adversely affecting the synthesis of plastoquinone crucial for normal plant growth and development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Baack EJ, Whitney KD, Rieseberg LH (2005) Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species. New Phytol 167:321–333

    Article  CAS  Google Scholar 

  • Bennetzen JL (2000) Mechanisms and rates of genome expansion and contraction in flowering plants. Genetica 115:251–269 29–36

    Google Scholar 

  • Bennetzen JL (2002) Transposable element contributions to plant gene and genome evolution. Plant Mol Biol 42:251–269

    Article  Google Scholar 

  • Bergmüller E, Porfirova S, Dörmann P (2003) Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase. Plant Mol Biol 52:1181–1190

    Article  PubMed  Google Scholar 

  • Bowen NJ, McDonald JF (2001) Drosophila euchromatic LTR-retrotransposons are much younger than the host species in which they reside. Genome Res 11:1527–1540

    Article  PubMed  CAS  Google Scholar 

  • Brown JWS (1996) Arabidopsis intron mutations and pre-mRNA splicing. Plant J 10:771–780

    Article  PubMed  CAS  Google Scholar 

  • Cheng S, Fockler C, Barnes W, Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci USA 91:5695–5699

    Article  PubMed  CAS  Google Scholar 

  • Cheng Z, Sattler S, Maeda H, Sakuragi Y, Bryant DA, DellaPenna D (2003) Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes. Plant Cell 15:2343–2356

    Article  PubMed  CAS  Google Scholar 

  • Collakova E, DellaPenna D (2001) Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp PCC 6803 and Arabidopsis. Plant Physiol 127:1113–1124

    Article  PubMed  CAS  Google Scholar 

  • Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S (2004) Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37:778–86

    Article  PubMed  CAS  Google Scholar 

  • Comai L, Henikoff S (2006) TILLING: practical single-nucleotide mutation discovery. Plant J 45:684–94

    Article  PubMed  CAS  Google Scholar 

  • Cook W, Miles D (1992) Nuclear mutations affecting plastoquinone accumulation in maize. Photosynth Res 31:99–111

    Article  CAS  Google Scholar 

  • Cui X, Hsiac AP, Liu F, Ashlockd DA, Wise RP, Schnable PS (2003) Alternative transcription initiation sites and polyadenylation sites are recruited during Mu suppression at the rf2a locus of maize. Genetics 163:685–698

    PubMed  CAS  Google Scholar 

  • Demurin Y (1993) Genetic variability of tocopherol composition in sunflower seeds. Helia 16:59–62

    Google Scholar 

  • Demurin Y, Skoric D, Karlovic D (1996) Genetic variability of tocopherol composition in sunflower seeds as a basis of breeding for improved oil quality. Plant Breed 115:33–36

    Article  CAS  Google Scholar 

  • Demurin Y, Efimenko SG, Peretyagina TM (2004) Genetic identification of tocopherol mutations in sunflower. Helia 27:113–116

    Article  Google Scholar 

  • Dolde D, Vlahakis C, Hazebroek J (1999) Tocopherols in breeding lines and effects of plant location, fatty acid composition, and temperature during development. J Am Oil Chem Soc 76:349–355

    Article  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  PubMed  CAS  Google Scholar 

  • Feschotte C, Jiang N, Wessler SR (2002) Plant transposable elements: where genetics meets genomics. Nature Rev Genetics 3:329–341

    Article  CAS  Google Scholar 

  • Fick GN, Zimmer DE, Kinman ML (1974) Registration of six sunflower parental lines. Crop Sci 14:912

    Article  Google Scholar 

  • Grandbastein AM, Spielman A, Caboche M (1989) Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature 337:376–380

    Article  Google Scholar 

  • Grandbastein AM (1998). Activation of plant retrotransposons under stress conditions. Trends Plant Sci. 3:181–187

    Article  Google Scholar 

  • Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–40

    PubMed  CAS  Google Scholar 

  • Grusak MA, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50:133–161

    Article  PubMed  CAS  Google Scholar 

  • Hass CG, Leonard SW, Miller JF, Slabaugh MB, Traber MG, Knapp SJ (2003) Genetics of tocopherol (vitamin E) composition mutants in sunflower. In: Abstract of Plant and Animal Genome Conference XI, San Diego, CA, USA, Jan 11–15, 2003 (http://wwwintl-pagorg/11/ abstracts/P7b_P821_XI.html)

  • Hass CG, Tang S, Leonard S, Traber M, Miller JF, Knapp SJ (2006) Three non-allelic epistatically interacting methyltransferase mutations produce novel tocopherol (vitamin E) profiles in sunflower. Theor Appl Genet (in press)

  • Hoffmann GR (1980) Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds. Mutat Res 75:63–129

    PubMed  CAS  Google Scholar 

  • Jiang N, Feschotte C, Zhang X, Wessler SR (2004) Using rice to understand the origin and amplification of miniature inverted repeat transposable elements (MITEs). Curr Opin Plant Biol 7:115–119

    Article  PubMed  CAS  Google Scholar 

  • Johns MA, Mottinger J, Freeling M (1985) A low copy number, copia-like transposon in maize. EMBO J 4:1093–1102

    PubMed  CAS  Google Scholar 

  • Joshi C, Chiang V (1998) Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol 37:663–674

    Article  PubMed  CAS  Google Scholar 

  • Kagan R, Clarke S (1994) Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch Biochem Biophys 310:417–427

    Article  PubMed  CAS  Google Scholar 

  • Kamal-Eldin A, Andersson R (1997) A multivariate study of the correlation between tocopherols composition content and fatty acid composition in vegetable oils. J Am Oil Chem Soc 74:375–380

    Article  CAS  Google Scholar 

  • Lal SK, Choi J-H, Shaw JR, Hannah LC (1999) A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation. Plant Physiol 121:411–418

    Article  PubMed  CAS  Google Scholar 

  • Lal SK, Giroux MJ, Brendel V, Vallejos CE, Hannah LC (2003) The maize genome contains a helitron insertion. Plant Cell 15:381–391

    Article  PubMed  CAS  Google Scholar 

  • Lal SK, Hannah LC (2005) Plant genomes: massive changes of the maize genome are caused by Helitrons. Heredity 95:421–2

    Article  PubMed  CAS  Google Scholar 

  • Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181

    Article  PubMed  CAS  Google Scholar 

  • Ma J, Devos KM, Bennetzen JL (2004) Analyses of LTR-retrotransposon structures reveal recent and rapid genomic DNA loss in rice. Genome Res 14:860–869

    Article  PubMed  CAS  Google Scholar 

  • Marchler-Bauer A, Bryant SH (2004) CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32:W327–W331

    Article  PubMed  CAS  Google Scholar 

  • Marillonnet S, Wessler SR (1997) Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing. Plant Cell 9:967–78

    Article  PubMed  CAS  Google Scholar 

  • Marillonnet S, Wessler SR (1998) Extreme structural heterogeneity among the members of a maize retrotransposon family. Genetics 150:1245–56

    PubMed  CAS  Google Scholar 

  • Michelmore RW, Paran I, Kesseli V (1991) Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88:9828–9832

    Article  PubMed  CAS  Google Scholar 

  • Miller JF (1997) Registration of cms HA89 (PEF1) cytoplasmic male-sterile, RPEF1 restorer, and two nuclear male-sterile (NMS373 and 377) sunflower genetic stocks. Crop Sci 37:1984

    Article  Google Scholar 

  • Motohashi R, Ito T, Kobayashi M, Taji T, Nagata N, Asami T, Yoshida S, Yamaguchi-Shinozaki K, Shinozaki K (2003) Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant. Plant J 34:719–731

    Article  PubMed  CAS  Google Scholar 

  • Perez-Vich B, Berry ST, Velasco L, Fernandez-Martinez JM, Gandhi S, Freeman C, Heesacker A, Knapp SJ, Leon AJ (2005) Molecular mapping of nuclear male sterility genes in sunflower. Crop Sci 45:1851–1857

    Article  CAS  Google Scholar 

  • Porfirova S, Bergmüller E, Tropf S, Lemke R, Dormann P (2002) Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis. Proc Natl Acad Sci USA 99:12495–12500

    Article  PubMed  CAS  Google Scholar 

  • Purugganan MD, Wessler SR (1994) Molecular evolution of magellan, a maize Ty3/gypsy-like retrotransposon. Proc Natl Acad Sci USA 91:11674–11678

    Article  PubMed  CAS  Google Scholar 

  • Roath WW, Miller JF, Gulya TJ (1981) Registration of RHA 801 sunflower germplasm. Crop Sci 21:479

    Article  Google Scholar 

  • Rocheford TR, Wong JC, Egesel CO, Lambert RJ (2002) Enhancement of vitamin E levels in corn. J Am Coll Nutr 21:191S–198S

    PubMed  CAS  Google Scholar 

  • Rozas J, Sanchez-Delbarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497

    Article  PubMed  CAS  Google Scholar 

  • SanMiguel P, Tikhonov A, Jin YK, Motchoulskaia N, Zakharov D, Melake-Berhan A, Springer PS, Edwards KJ, Lee M, Avramova Z, Bennetzen JL (1996) Nested retrotransposons in the intergenic regions of the maize genome. Science 274:765–768

    Article  PubMed  CAS  Google Scholar 

  • SanMiguel P, Gaut BS, Tikhonov A, Nakajima Y, Bennetzen JL (1998) The paleontology of intergene retrotransposons of maize. Nature Genet 20:43–45

    Article  PubMed  CAS  Google Scholar 

  • Santini S, Cavallini A, Natali L, Minelli S, Maggini F, Cionini PG (2002) Ty1/copia- and Ty3/ gypsy-like DNA sequences in Helianthus species. Chromosoma 111:192–120

    Article  PubMed  CAS  Google Scholar 

  • Sattler SE, Cahoon EB, Coughlan SJ, DellaPenna D (2003) Characterization of tocopherol cyclases from higher plants and cyanobacteria: evolutionary implications for tocopherol synthesis and function. Plant Physiol 132:2184–2195

    Article  PubMed  CAS  Google Scholar 

  • Sattler SE, Gillilanda LU, Magallanes-Lundbacka M, Pollard M, DellaPenna D (2004) Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16:1419–1432

    Article  PubMed  CAS  Google Scholar 

  • Sheppard AJ, Pennington JAT, Weihrauch JL (1993) Analysis and distribution of vitamin E in vegetable oils and foods. In: Packer L, Fuch J (eds) Vitamin E in health and disease. Marcel-Dekker, New York, pp 9–31

    Google Scholar 

  • Shintani DK, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2100

    Article  PubMed  CAS  Google Scholar 

  • Shintani DK, Cheng Z, DellaPenna D (2002) The role of 2-methyl-6-phytylbenzoquinone methyltransferase in determining tocopherol composition in Synechocystis sp. PCC6803. FEBS Lett. 511:1–5

    Article  PubMed  CAS  Google Scholar 

  • Song SU, Gerasimova T, Kurkulos M, Boeke JD, Corces VG (1994) An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus. Genes Dev 8:2046–2057

    Article  PubMed  CAS  Google Scholar 

  • Tang S, Yu JK, Slabaugh MB, Shintani DK, Knapp SJ (2002) Simple sequence repeat map of the sunflower genome. Theor Appl Genet 105:1124–1136

    Article  PubMed  CAS  Google Scholar 

  • Tang S, Kishore VK, Knapp SJ (2003) PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower. Theor Appl Genet 107:6–19

    PubMed  CAS  Google Scholar 

  • Tang S, Hass CG, Knapp SJ (2005) Candidate genes for mutations causing changes in tocopherol composition in sunflower. In: Abstract of Plant and Animal Genome Conference XIII, San Diego, CA, USA, Jan 15–19, 2005 (http://www.intl-pag.org/13/abstracts/PAG13_W071.html)

  • Tatusova TA, Madden TL (1999) Blast 2 sequences - a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174:247–250

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–30

    Article  PubMed  CAS  Google Scholar 

  • Van Eenennaam AL, Lincoln K, Durrett TP, Valentin HE, Shewmaker CK, Thorne GM, Jiang J, Baszis SR, Levering CK, Aasen ED, Hao M, Stein JC, Norris SR, Last RL (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell 15:3007–3019

    Article  PubMed  CAS  Google Scholar 

  • Varagona MJ, Purugganan M, Wessler SR (1992) Alternative splicing induced by insertion of retrotransposons into the maize waxy gene. Plant Cell 4:811–820

    Article  PubMed  CAS  Google Scholar 

  • Velasco L, Domínguez J, Fernández-Martínez JM (2004a) Registration of T589 and T2100 sunflower germplasms with modified tocopherol profiles. Crop Sci 44:362–363

    Article  Google Scholar 

  • Velasco L, Pérez-Vich B, Fernández-Martínez JM (2004b) Novel variation for the tocopherol profile in a sunflower created by mutagenesis and recombination. Plant Breed 123:490–492

    Article  CAS  Google Scholar 

  • Weil CF, Marillonnet S, Burr B, Wessler SR (1992) Changes in state of the Wx-m5 allele of maize are due to intragenic transposition of Ds. Genetics 130:75–85

    Google Scholar 

  • Wong JC, Lambert RT, Tadmor Y, Rocheford TR (2003) QTL associated with accumulation of tocopherols in maize. Crop Sci 43:2257–2266

    Article  CAS  Google Scholar 

  • Yu JK, Tang S, Slabaugh MB, Heesacker A, Cole G, Herring M, Soper J, Han F, Chu WC, Webb DM, Thompson L, Edwards KJ, Berry S, Leon AJ, Olungu C, Maes N, Knapp SJ (2003) Towards a saturated molecular genetic linkage map for cultivated sunflower. Crop Sci 43:367–387

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by funding to S.J.K. from the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service Plant Genome Program (Grant No. 2003-35300-15184), the Paul C. Berger Endowment at Oregon State University, the Georgia Research Alliance, and University of Georgia Research Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Steven J. Knapp.

Additional information

Communicated by C. Gebhardt

Shunxue Tang and Catherine G. Hass contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tang, S., Hass, C.G. & Knapp, S.J. Ty3/gypsy-like retrotransposon knockout of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase is non-lethal, uncovers a cryptic paralogous mutation, and produces novel tocopherol (vitamin E) profiles in sunflower. Theor Appl Genet 113, 783–799 (2006). https://doi.org/10.1007/s00122-006-0321-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00122-006-0321-3

Keywords

Navigation