Elevation of the provitamin A content of transgenic tomato plants

Abstract

Tomato products are the principal dietary sources of lycopene and major source of β-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the β-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Carotenoid biosynthesis showing the diversity of desaturases.
Figure 2: Uniform distribution of CRTI protein among different fruit tissues.
Figure 3: Northern blot analysis of crtI primary transformants.
Figure 4: Inheritance and expression of crtI in 20 progeny plants.

References

  1. 1

    Mayne, S.T. β-Carotene, carotenoids and disease prevention in humans. FASEB J. 10, 690–701 (1996).

  2. 2

    Humphrey, J.H., West Jr. K.P. & Sommer, A. Vitamin A deficiency and attributable mortality among under 5 year olds, WHO Bull. 70, 225–232 (1992).

  3. 3

    Suber, A. et al. 5 A day for better health! A baseline study of American's fruit and vegetable consumption. (Natl. Cancer Inst., Washington, DC; 1992).

  4. 4

    Tomato News January 1999 issue. URL: http://www.Tomato.org

  5. 5

    Sandmann, G. Carotenoid biosynthesis in microorganisms and plants. Eur. J. Biochem. 223, 7–24 (1994).

  6. 6

    Fraser, P.D. et al. Expression in E. coli, purification and reactivation of the recombinant Erwinia uredovora phytoene desaturase. J. Biol. Chem. 267, 19891–19895 (1992).

  7. 7

    Misawa, N. et al. Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtI in transgenic plants showing an increase of β-carotene biosynthesis activity and resistance to the bleaching herbicide Norflurazon. Plant J. 4, 833–840 (1993).

  8. 8

    Misawa, N. et al. Expression of an Erwinia uredovora phytoene desaturase gene not only confers multiple resistance to herbicides interfering with carotenoid biosynthesis but also alters xanthophyll metabolism in transgenic plants. Plant J. 6, 481–489 (1994).

  9. 9

    Fray, R.G. et al. Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway. Plant J. 8, 693–701 (1995).

  10. 10

    Bird, C.R. et al. The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Mol. Biol. 11, 651–662 (1988).

  11. 11

    McCormick, D.B., In Tietz fundamentals of clinical chemistry. (eds Burtis, C.A. & Ashwood, E.R.) 469–483 (W.B. Saunders, London; 1995).

  12. 12

    von den Berg, H. Carotenoid interactions. Nutr. Rev. 57, 1–10 (1999).

  13. 13

    Fraser, P.D., Truesdale, M., Bird, C.R., Schuch, W. & Bramley, P.M. Carotenoid biosynthesis during tomato fruit development. Evidence for fruit-specific gene expression. Plant Physiol. 105, 405–413 (1994).

  14. 14

    Sambrook, J., Fritsch, E.F. & Maniatis T. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, New York, NY; 1989).

  15. 15

    Kuntz, M. et al. Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening. Plant J. 2, 25–34 (1992).

  16. 16

    Fraser, P.D., Kiano, J.W., Truesdale, M.R., Schuch, W. & Bramley, P.M. Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit. Plant Mol. Biol.. 40, 687–698 (1999).

  17. 17

    Lichtenthaler, H. K. & Wellburn, A.R. Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592 (1983).

  18. 18

    Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685 (1970).

  19. 19

    Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.. 247, 942–950 (1976).

  20. 20

    Wessel, D. & Flügge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

  21. 21

    Fraser, P.D., De la Rivas, J., Mackenzie, A. & Bramley, P.M. Phycomyces blakesleeanus carB mutants: Their use in assays of phytoene desaturase. Phytochemistry 30, 3971–3976 (1991).

  22. 22

    Ye X. et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endopserm. Science 287, 303–305 (2000).

Download references

Acknowledgements

Financial support was principally from the European Community BIOTECH Programme (#B102 CT-930400, as part of the project of Technological Priority 1993–1996). We thank Rachel Drake for helpful discussions. Zeneca Agrochemicals is gratefully acknowledged for the provision of labatory and greenhouse space. We thank Karen Bacon for maintenance of the plants.

Author information

Correspondence to Peter M. Bramley.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Römer, S., Fraser, P., Kiano, J. et al. Elevation of the provitamin A content of transgenic tomato plants. Nat Biotechnol 18, 666–669 (2000). https://doi.org/10.1038/76523

Download citation

Further reading