Plant Molecular Biology

, Volume 52, Issue 6, pp 1181–1190 | Cite as

Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

  • Eveline Bergmüller
  • Svetlana Porfirova
  • Peter Dörmann

Abstract

Alpha-tocopherol (vitamin E) is synthesized from γ-tocopherol in chloroplasts by γ-tocopherol methyltransferase (γ-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of α-tocopherol, but are low in γ-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene γ-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allel (vte4-2) carrying a T-DNA insertion in the coding sequence of γ-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of γ-tocopherol accumulated, whereas α-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the γ-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of α-tocopherol and γ-tocopherol increased in wild type, and γ-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that α-tocopherol can be replaced by γ-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.

lipid mutant oxidative stress tocopherol vitamin E 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bechtold, N., Ellis, J. and Pelletier, G. 1993. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. Paris Life Sci. 316: 1194–1199.Google Scholar
  2. Bell, C.J. and Ecker, J.R. 1994. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19: 137–144.Google Scholar
  3. Bent, A.F., Kunkel, B.N., Dahlbeck, D., Brown, K.L., Schmidt R., Giraudat, J., Leung, J. and Staskawicz, B.J. 1994. RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance gene. Science 265: 1856–1860.Google Scholar
  4. Browse, J., McCourt, P.J. and Somerville, C.R. 1986. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal. Biochem. 152: 141–145.Google Scholar
  5. Collakova, E. and DellaPenna, D. 2001. Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 127: 1113–1124.Google Scholar
  6. Collakova, E. and DellaPenna, D. 2003. Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol. 131: 632–642.Google Scholar
  7. DellaPenna, D. 1999. Nutritional genomics: manipulating plant micronutrients to improve human health. Science 285: 375–379.Google Scholar
  8. Esterbauer, H. and Cheeseman, K.H. 1990. Determination of aldehydic lipid peroxidation products: malondehyde and 4-hydroxynonenal. Meth. Enzymol. 186: 407–421.Google Scholar
  9. Evans, H.M. and Bishop, K.S. 1922. The relations between fertility and nutrition. I. The ovulation rhythm in the rat on a standard nutritional regime. II. The ovulation rhythm in the rat on inadequate nutritional regimes. J. Metabol. Res. 1: 319–355.Google Scholar
  10. Fiehn, O., Kopka, J., Trethewey, R.N. and Willmitzer, L. 2000. Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry. Anal. Chem. 72: 3573–3580.Google Scholar
  11. Fryer, M.J. 1992. The antioxidant effects of thylakoid vitamin E (α-tocopherol) Plant Cell Environ. 15: 381–392.Google Scholar
  12. Fukuzawa, K., Tokumura, A., Ouchi, S. and Tsukatani, H. 1982. Antioxidant activities of tocopherols on Fe2+-ascorbate-induced lipid peroxidation in lecithin liposomes. Lipids 17: 511–513.Google Scholar
  13. Garcia, I., Rodgers, M., Lenne, C., Rolland, A., Sailland, A. and Matringe, M. 1997. Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA. Biochem. J. 325: 761–769.Google Scholar
  14. Grau, A. and Ortiz, A. 1998. Dissimilar protection of tocopherol isomers against membrane hydrolysis by phospholipase A2. Chem. Phys. Lipids 91: 109–118.Google Scholar
  15. Grusak, M.A. and 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.Google Scholar
  16. Hanley, B.A. and Schuler, M.A. 1988. Plant intron sequences: evidence for distinct groups of introns. Nucl. Acids. Res. 16: 7159–7176.Google Scholar
  17. Henry, A., Powls, R. and Pennock, J.F. 1987. Intermediates of tocopherol biosynthesis in the unicellular alga Scenedesmus obliquus. Biochem. J. 242: 367–373.Google Scholar
  18. Herbers, K., Badur, R., Kunze, I. and Geiger, M. 2001. Identification and overexpression of a DNA sequence coding for 2-methyl-6-phytylhydroquinone-methyltransferase in plants. International Patent WO 01/04330.Google Scholar
  19. Höfgen, R. and Willmitzer, L. 1990. Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solanum tuberosum). Plant Sci. 66: 221–230.Google Scholar
  20. Kagan, V.E. and Quinn, P.J. 1988. The interaction of α-tocopherol and homologues with shorter hydrocarbon chains with phospholipids bilayer dispersions. A fluorescence probe study. Eur. J. Biochem. 171: 661–668.Google Scholar
  21. Kamal-Eldin, A. and Appelqvist, L.A. 1996. The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31: 671–701.Google Scholar
  22. Koch, M., Lemke, R., Heise, K.-P. and Mock, H.-P. 2003. Characterization of γ-tocopherol methyltransferase from Capsicum annuum L. and Arabidopsis thaliana. Eur. J. Biochem. 270: 84–92.Google Scholar
  23. Konieczny, A. and Ausubel, F.M. 1993. A procedure for mapping Arabidopsis mutations using co-dominant ecotype specific PCRbased markers. Plant J. 4: 403–410.Google Scholar
  24. Landry, L.G., Chapple, C.C.S. and Last, R.L. 1995. Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol. 109: 1159–1166.Google Scholar
  25. Lichtenthaler, H.K. 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth. Enzymol. 148: 350–382.Google Scholar
  26. Lichtenthaler, H., Prenzel, U., Douce, R. and Joyard, J. 1981. Localization of prenylquinones in the envelope of spinach chloroplasts. Biochim. Biophys. Acta 641: 99–105.Google Scholar
  27. McKersie, B.D., Hoekstra, F.A. and Krieg, L.C. 1990. Differences in the susceptibility of plant membrane lipids to peroxidation. Biochim. Biophys. Acta 1030: 119–126.Google Scholar
  28. Noctor, G. and Foyer, C.H. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 249–279.Google Scholar
  29. Norris, S.R., Barrette, T.R. and DellaPenna, D. 1995. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 7: 2139–2149.Google Scholar
  30. Norris, S.R., Shen, X. and DellaPenna, D. 1998. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. Plant Physiol. 117: 1317–1323.Google Scholar
  31. Pennell, R.I. and Lamb, C. 1997. Programmed cell death in plants. Plant Cell 9: 1157–1168.Google Scholar
  32. Porfirova, S., Bergmüller, E., Tropf, S., Lemke, R. and Dörmann, 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.Google Scholar
  33. Reeves, P.H. and Coupland, G. 2001. Analysis of flowering time control in Arabidopsis by comparison of double and triple mutants. Plant Physiol. 126: 1085–1091.Google Scholar
  34. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview, NY.Google Scholar
  35. Schreiber, U., Schliwa, U. and Bilger, W. 1986. Continous recording of photochemical and nonphotochemical quenching with a new type of modulation fluorometer. Photosynth. Res. 10: 51–62.Google Scholar
  36. Sessions, A., Burke, E., Presting, G., Aux, G., McElver, J., Patton, D., Dietrich, B., Ho, P., Bacwaden, J., Ko, C., Clarke, J.D., Cotton, D., Bullis, D., Snell, J., Miguel, T., Hutchison, D., Kimmerly, B., Mitzel, T., Katagiri, F., Glazebrook, J., Law, M. and Goff, S.A. 2002. A high-throughput Arabidopsis reverse genetics system. Plant Cell 14: 2986–2994.Google Scholar
  37. Shintani, D. and DellaPenna, D. 1998. Elevating the vitamin E content of plants through metabolic engineering. Science 282: 2098–2100.Google Scholar
  38. Soll, J., Schultz, G., Joyard, J., Douce, R. and Block, M.A. 1985. Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch. Biochem. Biophys. 238: 290–299.Google Scholar
  39. Thompson, J.N. and Hatina, G. 1979. Determination of tocopherols and tocotrienols in foods and tissues by high performance liquid chromatography. J. Liquid Chromat. 2: 327–344.Google Scholar
  40. Tsegaye, Y., Shintani, D.K. and DellaPenna, D. 2002. Overexpression of the enzyme p-hydroxyphenolpyruate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis. Plant Physiol. Biochem. 40: 913–920.Google Scholar
  41. Weiner, H., Stitt, M. and Heldt, H.W. 1987. Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim. Biophys. Acta 893: 13–21.Google Scholar
  42. Whistance, G.R. and Thelfall, D.R. 1970. Biosynthesis of phytoquinones. Homogentisic acid: a precursor of plastoquinones, tocopherols and α-tocopherolquinone in higher plants, green algae and blue-green algae.Biochem. J. 117: 593–600.Google Scholar
  43. Young, A.J. 1991. The photoprotective role of carotenoids in higher plants. Physiol. Plant. 83: 702–708.Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Eveline Bergmüller
    • 1
  • Svetlana Porfirova
    • 1
  • Peter Dörmann
    • 1
  1. 1.Department of Lothar WillmitzerMax Planck Institute of Molecular Plant PhysiologyGolmGermany

Personalised recommendations