Skip to main content
SpringerLink
Log in

Stable expression of 1Dx5 and 1Dy10 high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We generated and characterized transgenic rye synthesizing substantial amounts of high-molecular-weight glutenin subunits (HMW-GS) from wheat. The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the HMW-GS. Rye flour has poor bread-making quality, despite the extensive sequence and structure similarities of wheat and rye HMW-GS. The HMW-GS 1Dx5 and 1Dy10 genes from wheat, known to be associated with good bread-making quality were introduced into a homozygous rye inbred line by the biolistic gene transfer. The transgenic plants, regenerated from immature embryo derived callus cultures were normal, fertile, and transmitted the transgenes stably to the sexual progeny, as shown by Southern blot and SDS-PAGE analysis. Flour proteins were extracted by means of a modified Osborne fractionation from wildtype (L22) as well as transgenic rye expressing 1Dy10 (L26) or 1Dx5 and 1Dy10 (L8) and were quantified by RP-HPLC and GP-HPLC. The amount of transgenic HMW-GS in homozygous rye seeds represented 5.1% (L26) or 16.3% (L8) of the total extracted protein and 17% (L26) or 29% (L8) of the extracted glutelin fraction. The amount of polymerized glutelins was significantly increased in transgenic rye (L26) and more than tripled in transgenic rye (L8) compared to wildtype (L22). Gel permeation HPLC of the un-polymerized fractions revealed that the transgenic rye flours contained a significantly lower proportion of alcohol-soluble oligomeric proteins compared with the non-transgenic flour. The quantitative data indicate that the expression of wheat HMW-GS in rye leads to a high degree of polymerization of transgenic and native storage proteins, probably by formation of intermolecular disulfide bonds. Even γ-40k secalins, which occur in non-transgenic rye as monomers, are incorporated into these polymeric structures. The combination 1Dx5 + 1Dy10 showed stronger effects than 1Dy10 alone. Our results are the first example of genetic engineering to significantly alter the polymerization and composition of storage proteins in rye. This may be an important step towards improving bread-making properties of rye whilst conserving its superior stress resistance.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altpeter, F., Vasil, V., Srivastava, V. and Vasil, I.K. 1996. Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nature Biotechnol. 14: 1155–1159.

    Google Scholar 

  • Altpeter, F. and Xu, J. 2000. Rapid production of transgenic turfgrass (Festuca rubra L.) plants. J. Plant Physiol. 157: 441–448.

    Google Scholar 

  • Barro, F., Rooke, L., Bekes, F., Gras, P., Tatham, A.S., Fido, R., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. 1997. Transformation of wheat with high-molecular-weight subunit genes results in improved functional properties. Nature Biotechnol. 15: 1295–1299.

    Google Scholar 

  • Blechl, A.E. and Anderson, O.D. 1996. Expression of a novel high molecular weight glutenin subunit gene in transgenic wheat. Nature Biotechnol. 14: 875–879.

    Google Scholar 

  • Blechl, A.E., Le, H.Q. and Anderson, O.D. 1998. Engineering changes in wheat flour by genetic transformation. J. Plant Physiol. 152: 703–707.

    Google Scholar 

  • Bushuk W. 2001. RYE: Production, Chemistry, and Technology, Second Edition AACC, St Paul, MN.

    Google Scholar 

  • Castillo, A.M., Vasil, V. and Vasil, I.K. 1994. Rapid production of fertile transgenic plants of rye (Secale cereale L.). Bio/Technology 12: 1366–1371.

    Google Scholar 

  • Dellaporta, S.L., Wood, J. and Hicks, J.B. 1983. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 4: 19–21.

    Google Scholar 

  • De Bustos, A. and Jouve, N. 2003. Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale. Theor. Appl. Genet. 107: 74–83.

    PubMed  Google Scholar 

  • De Bustos, A., Rubio, P. and Jouve, N. 2001. Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat. Theor. Appl. Genet. 103: 733–742.

    Google Scholar 

  • D'Ovidio, R. and Anderson, O.D. 1994. PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus. Theor. Appl. Genet. 88: 175–180.

    Google Scholar 

  • Gellrich, C., Schieberle, P. and Wieser, H. 2003. Biochemical characterization and quantification of storage protein (secalin) types in rye flour. Cereal Chem. 80: 102–109.

    Google Scholar 

  • He, G.Y., Rooke, L., Steele, S., Békés, F., Gras, P., Tatham, A.S., Fido, R., Barcelo, P., Shewry, P.R. and Lazzeri, P. 1999. Transformation of pasta wheat (Triticum turgidum L. var. durum L.) with HMW glutenin subunit genes and modification of dough functionality. Mole. Breeding 5: 377–386.

    Google Scholar 

  • Kipp, B., Belitz, H.-D., Seilmeier, W. and Wieser, H. 1996. Comparative studies of high Mr subunits of rye and wheat. I. Isolation and biochemical characterisation and effects on gluten extensibility. J. Cereal Sci. 23: 227–234.

    Google Scholar 

  • Kipp, B. and Wieser H. 1999. Comparative Studies of High Mr Subunits of Rye and Wheat. II. Partial Amino Acid Sequences. J. Cereal Sci. 30: 303–313.

    Google Scholar 

  • Köhler, P. and Wieser, H. 2000. Comparative studies of high Mr subunits of rye and wheat. III. Localisation of cysteine residues. J. Cereal Sci. 32: 189–197.

    Google Scholar 

  • Lukow, O.M., Payne, P.I. and Tkachuk, R. 1989. The HMW glutenin subunit composition of Canadian wheat cultivars and their association with bread-making quality. J. Sci. Food. Agric. 46: 451–460.

    Google Scholar 

  • Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant. Physiol. 15: 473–497.

    Google Scholar 

  • Payne, P.I., Nightingale, M.A., Krattiger, A.F. and Holt, L.M. 1987. The relation between HMW glutenin subunit composition and the bread-making quality of British-grown wheat varieties. J. Sci. Food Agric. 40: 51–65.

    Google Scholar 

  • Popelka, J.C. and Altpeter, F. 2001. Interactions between genotypes and culture media components for improved in vitro response of rye (Secale cereale L.) inbred lines. Plant Cell Rep. 20: 575–582.

    Google Scholar 

  • Popelka, J.C. and Altpeter, F. 2003a. Evaluation of rye (Secale cereale L.) inbred lines and their crosses for tissue culture response and stable genetic transformation of homozygous rye inbred line L22 by biolistic gene transfer. Theor. Appl. Genet. 107: 583–590.

    PubMed  Google Scholar 

  • Popelka, J.C. and Altpeter, F. 2003b. Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.). Mol. Breeding 11: 203–211.

    Google Scholar 

  • Rooke, L., Barro, F., Tatham, A.S., Fido, R., Steele, S., Békés, F., Gras, P., Martin, A., Lazzeri, P.A., Shewry, P.R. and Barcelo, P. 2000. Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes. Theor. Appl. Genet. 99: 851–858.

    Google Scholar 

  • Sanford, J.C., De Vit, M.J., Russel, J.A., Smith, F.D., Harpening, P.R., Roy, M.K. and Johnston, S.A. 1991. An Improved, helium-driven biolistic device. Technique 3: 3–16.

    Google Scholar 

  • Shewry, P.R., Halford, N.G. and Tatham, A.S. 1992. High molecular weight subunits of wheat glutenin. J. Cereal Sci. 15: 105–120.

    Google Scholar 

  • Shewry, P.R., Miflin, B.J. and Kasarda, D.D. 1984. The structural and evolutionary relationships of the prolamin storage proteins of barley, rye and wheat. Phil. Trans. R. Soc. Lond. B 304: 297–308.

    Google Scholar 

  • Shewry, P.R., Popineau, Y., Lafiandra, D. and Belton, P. 2001. Wheat glutenin subunits and dough elasticity: findings of the EUROWHEAT project. Trends Food Sci. Tech. 11: 433–441.

    Google Scholar 

  • Vasil, I.K., Bean, S., Zhao, J., McClusskey, P., Lookhart, G., Zhao, H.-P., Altpeter, F. and Vasil, V. 2001. Evaluation of baking properties and gluten protein composition of field grown transgenic wheat lines expressing high molecular weight glutenin gene 1Ax1. J. Plant Physiol. 158: 521–528.

    Google Scholar 

  • Wieser, H., Antes, S. and Seilmeier, W. 1998. Quantitative determination of gluten protein types in wheat flour by reversed-phase high-performance liquid chromatography. Cereal Chem. 75: 644–650.

    Google Scholar 

  • Wieser, H. and Zimmermann, G. 2000. Importance of amounts and proportions of high molecular weight subunits of glutenin for wheat quality. Eur. Food Res. Technol. 210: 324–330.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Agronomy Department, Laboratory of Molecular Plant Physiology, University of Florida – IFAS, 2191 McCarty Hall, Gainesville, FL, USA

    Fredy Altpeter

  2. CSIRO Plant Industry, ACT, Australia

    Carlos Popelka Juan

  3. Deutsche Forschungsanstalt fuer Lebensmittelchemie (DFA), Lichtenbergstr. 4, Germany

    Herbert Wieser

Authors
  1. Fredy Altpeter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carlos Popelka Juan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herbert Wieser
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Altpeter, F., Juan, C.P. & Wieser, H. Stable expression of 1Dx5 and 1Dy10 high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour. Plant Mol Biol 54, 783–792 (2004). https://doi.org/10.1007/s11103-004-0122-5

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-004-0122-5

Search

Navigation