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Iron fortification of rice seed by the soybean ferritin gene

Abstract

To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L. cv. Kita-ake) by Agrobacterium-mediated transformation. The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis. Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing. The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.

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Figure 1: (A) Construction of GluB-1 promoter—soybean ferritin chimeric gene in the pGPTV-35S-bar binary vector.
Figure 2: Western blot analysis of soybean ferritin protein in the transgenic rice seeds.
Figure 3: Immunological tissue printing of a seed from transgenic rice expressing soybean ferritin cDNA.
Figure 4: Comparison of iron content in transgenic rice seeds expressing soybean ferritin.
Figure 5: Distribution of iron in the rice seeds transformed with soybean ferritin cDNA.

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References

  1. World Health Organization (WHO). National Strategies for Overcoming Micronutrient Malnutrition. Document A45/3. (WHO, Geneva, Switzerland, 1992).

  2. Craig, W.J. Iron status of vegetarians. Am. J. Clin. Nutr. 59, 1233S–1237S (1994).

    Article  CAS  Google Scholar 

  3. Yip, R. Iron deficiency, contemporary scientific issues and international programmatic approaches. J. Nutr. 124, 1479S– 1490S (1994).

    Article  CAS  Google Scholar 

  4. Anonymous. in Standard Tables of Food Composition (ed. Investigation Committee of Food Resources, Science and Technology Agency, Japan, 1992).

  5. Gillooly, M. et al. The effect of organic acids, phytates and polyphenols on the absorption of iron from vegetables. Br. J. Nutr. 49, 331–342 (1983).

    Article  CAS  Google Scholar 

  6. Inoue, K., Tashima, K., Sanada, K. & Yokota, H. Production of iron-enriched leaf vegetables using a soaking method. Jap. J. Soil Sci. Plant Nutr. 66, 527–534 ( 1995).

    CAS  Google Scholar 

  7. Theil, E.C. Ferritin: structure, gene regulation, and cellular function in animals, plants and microorganisms. Annu. Rev. Biochem. 56, 289–315 (1987).

    Article  CAS  Google Scholar 

  8. Andrews, S. et al. Structure, function and evolution of ferritins. J. Inorg. Chem. 47, 161–174 ( 1992).

    CAS  Google Scholar 

  9. Ragland, M. et al. Evidence for a conservation of ferritin sequences among plants and animals and for a transit peptide in soybean. J. Biol. Chem. 265, 18339–18344 (1990).

    CAS  PubMed  Google Scholar 

  10. Spence, M.J., Henzl, M.T. & Lammers, P.J. The structure of a Phaseolus vulgaris cDNA encoding the iron storage protein ferritin. Plant Mol Biol. 17, 499–504 (1991).

    Article  CAS  Google Scholar 

  11. Lobréaux, S., Yewdall, S., Briat, J.F. & Harrison, P.M. Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum ) ferritin. Biochem J. 288, 931– 939 (1992).

    Article  Google Scholar 

  12. Lobréaux, S., Massenet, O. & Briat, J.F. Iron induces ferritin synthesis in maize plantlets. Plant Mol Biol. 19, 563– 575 (1992).

    Article  Google Scholar 

  13. Theil, E.C. Regulation of ferritin and transferrin receptor mRNAs. J. Biol. Chem. 265, 4771–4774 ( 1990).

    CAS  PubMed  Google Scholar 

  14. Van der Mark, F., Bienfait, F. & van den Ende, H. Variable amounts of translatable ferritin mRNA in bean leaves with various iron contents. Biochem. Biophys. Res. Commun. 115, 463–469 ( 1983).

    Article  CAS  Google Scholar 

  15. Lescure, A.M. et al. Ferritin gene transcription is regulated by iron in soybean cell cultures. Proc. Natl. Acad. Sci. USA 88, 8222–8226 (1991).

    Article  CAS  Google Scholar 

  16. Lobréaux, S. & Briat, J.F. Ferritin accumulation and degradation in different organs of pea (Pisum sativum) during development. Biochem. J. 274, 601–606 (1991).

    Article  Google Scholar 

  17. Ragland, M. & Theil, E.C. Ferritin (mRNA. Protein) and iron concentration during soybean nodule development. Plant. Mol. Biol . 21, 555–560 ( 1993).

    Article  CAS  Google Scholar 

  18. Theil, E.C., Burton, J.W. & Beard J.L. A sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control. Eur. J. Clin. Nutr. 51, S28– S31 (1997).

    PubMed  Google Scholar 

  19. Beard, J.L., Burton, J.W. & Theil E.C. Purified ferritin and soybean meal can be sources of iron for treating iron deficiency in rats. J. Nutr. 126, 154–160 (1996).

    Article  CAS  Google Scholar 

  20. Takaiwa, F., Oono, K., Wing, D. & Kato, A. Sequences of three members and expression of a new major subfamily of glutelin gene from rice. Plant Mol. Biol. 17, 875– 885 (1991).

    Article  CAS  Google Scholar 

  21. Proudhon, D., Briat, J.F. & Lescure, A.M. Iron induction of ferritin synthesis in soybean cell suspensions. >Plant Physiol. 90, 586– 590 (1989).

    Article  CAS  Google Scholar 

  22. Kimata, Y. & Theil, E.C. Posttranscriptional regulation of ferritin during nodule development in soybean. Plant Physiol. 104, 263–270 (1994).

    Article  CAS  Google Scholar 

  23. Van Wuytswinkel, O., Savino, G. & Briat, J.F. Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro. Biochem. J. 305, 253–261 ( 1995).

    Article  CAS  Google Scholar 

  24. Van Wuytswinkel, O. & Briat, J.F. Conformational changes and in vitro core-formation modifications induced by site-directed mutagenesis of the specific N-terminus of pea seed ferritin. Biochem. J. 305, 959–965 ( 1995).

    Article  CAS  Google Scholar 

  25. Sturm, A., Voelker, T.A., Herman, E.M. & Chrispeels, M.J. Correct glycosylation, golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemaggletinin in transgenic tobacco. Planta. 175, 170–183 ( 1988).

    Article  CAS  Google Scholar 

  26. Ohtani, T., Wallace, J.C., Thompson, G.A., Galili, G. & Larkins, B.A. Normal and lysine containing zeins are unstable in transgenic tobaccoseeds. Plant Mol. Biol. 16, 117–128 (1990).

    Article  Google Scholar 

  27. Takaiwa, F. et al. High level accumulation of soybean glycinin in vacuole-derived protein bodies in the endosperm tissue of transgenic tobacco seed. Plant Sci. 111, 39–49 ( 1995).

    Article  CAS  Google Scholar 

  28. Takaiwa, F. et al. Characterization of common cis-regulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family. Plant Mol. Biol. 30, 1207– 1221 (1996).

    Article  CAS  Google Scholar 

  29. Wu, C.-Y., Suzuki, A., Washida, H. & Takaiwa, F. The GCN4 motif in a rice glutelin gene is essential for endosperm-specific expression and is activated by Opaque-2 in transgenic rice plants. Plant J. 14, 673–683 (1998).

    Article  CAS  Google Scholar 

  30. Utsumi, S. et al. Synthesis, processing and accumulation of modified glycinins of soybean in the seeds, leaves and stems of transgenic tobacco. Plant Sci. 92, 191–202 (1993).

    Article  CAS  Google Scholar 

  31. Shirsat, A.H., Wilford, N. & Croy, R.R.D. 1989.Gene copy number and levels of expression in transgenic plants of a seed specific gene. Plant Sci. 61, 75–80.

    Article  CAS  Google Scholar 

  32. Goto, F., Yoshihara, T. & Saiki, H. Iron accumulation in tobacco plants expressing soyabean ferritin gene. Transgenic Res. 7, 173– 180 (1998).

    Article  CAS  Google Scholar 

  33. Delhaize, E. A metal-accumulator mutant of Arabidopsis thaliana. Plant Physiol. 111, 849–855 ( 1996).

    Article  CAS  Google Scholar 

  34. Becker, D., Kemper, E., Schell, J. & Masterson, R. New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol. Biol. 20, 1195– 1197 (1992).

    Article  CAS  Google Scholar 

  35. Chu, C., Wang, C. & Sun, C. Establishment of an efficient medium for anther culture of rice through comparative experiment on the nitrogen sources. Scientia Sinica 18: 659-668 (1975).

  36. Murashige, T. & Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol. Plantarum. 15, 473–497 ( 1962).

    Article  CAS  Google Scholar 

  37. Shure, M., Wessler, S. & Fedoroff, N. Molecular identification and isolation of Waxy locus in maize. Cell 35, 225– 233 (1983).

    Article  CAS  Google Scholar 

  38. Enger-Blum, G., Meier, M., Frank, J. & Müller, G.A. Reduction of background problems in non-radioactive northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal. Biochem. 210, 235–244 (1993).

    Article  Google Scholar 

  39. Bradfold, M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 ( 1976).

    Article  Google Scholar 

  40. Sambrook, J., Fritsch, E.F. & Maniatis, T. in Molecular Cloning 2nd ed.(eds. Ford, N. & Nolan, C.) 18.67–75. (Cold Spring Harbor Laboratory Press, New York, 1989).

    Google Scholar 

  41. Kamachi, K., Yamaya, T., Hayakawa, T., Mae, T. & Ojima, K. Vascular bundle–specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol. 99, 1481– 1486 (1992).

    Article  CAS  Google Scholar 

  42. Anonymous. in Standard Methods for the Examination of Water and Wastewater. 18th edn (eds Greenberg, A.E., Clesceri, L.S., & Eaton, A.D.) 3-66–3–68 (American Public Health Association, Washington, DC, 1992).

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Acknowledgements

We wish to express our gratitude to J. Guerin, CSIRO (Commonwealth Scientific and Industrial Research Organization) for critically reading this paper and D. Becker (Max-Planck Institute) for providing the binary vector pGPTV-bar. We also thank Ms. Kusaka, Ms. Inoue, Ms. Miyake, and Ms. Tsunokawa (CRIEPI) for their technical assistance. This research was supported, in part, by research grants from the Enhancement Center-of-Excellence, the Special Coordination Funds for promoting the Science and Technology and from Bio-oriented Technology Research Advancement Institute (PRO-BRAIN) to F.T.

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Correspondence to Toshihiro Yoshihara or Fumio Takaiwa.

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Goto, F., Yoshihara, T., Shigemoto, N. et al. Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17, 282–286 (1999). https://doi.org/10.1038/7029

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