Modified Bean Seed Protein Phaseolin Did Not Accumulate Stably in Transgenic Tobacco Seeds after Methionine Enhancement Mutations


The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three-dimensional structure of protein, de novo design principles and genetic information. Amino acid substitution and loop insertion were targeted to the interior and exterior, respectively, of the protein’s β-barrels. First, we introduced the methionine enhancement mutations into phaseolin cDNA, expressed cDNA in Escherichia coli and purified monomeric non-glycosylated proteins. Biophysical analysis of E. coli-expressed proteins demonstrated a similar structural stability of wild-type and mutant phaseolin monomers. Here, we attempted to test the structural stability of the methionine-enhanced phaseolin by introducing phaseolin cDNA to tobacco via Agrobacterium tumefaciens-mediated transformation of leaf disks, regenerating transgenic tobacco plants, and examining the accumulation of phaseolin protein in mature transgenic tobacco seeds. We used seven constructs containing different extents of methionine enhancement, ranging from the original 3 to maximum 33 methionines per 397 amino acid residues. ELISA analyses indicated that the methionine-enhanced phaseolins did not accumulate as stably in mature transgenic tobacco seeds as the wild-type phaseolin. It seems likely that the methionine-enhanced phaseolin proteins were under the stringent scrutiny of the protein quality control mechanism in the endoplasmic reticulum (ER), Golgi complex and/or vacuolar protein bodies. The protein degradation is probably to occur in the vacuolar protein bodies due to the instability of the trimer assembly caused by the methionine enhancement mutations targeting either amino-acids substitutions or/and loop insertions to the interior β-sheets and tum/loop regions, respectively, of N- and C-barrel structures.

Share and Cite:

Lasserre, E. , Ko, T. , Dyer, J. and Murai, N. (2015) Modified Bean Seed Protein Phaseolin Did Not Accumulate Stably in Transgenic Tobacco Seeds after Methionine Enhancement Mutations. American Journal of Plant Sciences, 6, 640-650. doi: 10.4236/ajps.2015.65069.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Loesch Jr, P.J., Foley, D.C. and Cox, D.F. (1976) Comparative Resistance of opaque-2 and Normal Inbred Lines of Maize to Ear-Rotting Pathogens. Crop Science, 16, 841-842.
[2] Delaney, D.E. and Bliss, F.A. (1991) Selection for Increased Percentage Phaseolin in Common Bean. 1. Comparison of Selection for Seed Protein Alleles and S1 Family Recurrent Selection. Theoretical Applied Genetics, 81, 301-305.
[3] Dyer, J.M., Nelson, J.W. and Murai, N. (1993) Strategies for Selecting Mutation Sites for Methionine Enhancement in the Bean Seed Storage Protein Phaseolin. Journal Protein Chemistry, 12, 545-560.
[4] Dyer, J.M., Nelson, J.W. and Murai, N. (1995) Extensive Modifications for Methionine Enhancement in the β-Barrels Do Not Alter the Structural Stability of the Bean Storage Protein Phaseolin. Journal Protein Chemistry, 14, 665-677.
[5] Sun, S.M., Mutschler, M.A., Bliss, F.A. and Hall, T.C. (1978) Protein Synthesis and Accumulation in Bean Cotyledons during Growth. Plant Physiology, 61, 918-923.
[6] Bellini, R. and Chrispeels, M.J. (1978) Characterization and Subcellular Localization of Vicilin and Phytohemagglutinin, the Two Major Reserve Proteins of Phaseolus vulgaris L. Planta, 142, 291-298.
[7] Talbot, D.R., Adang, M.J., Slightom, J.L. and Hall, T.C. (1984) Size and Organization of a Multigene Family Encoding phaseolin, the Major Seed Storage Protein of Phaseolus vulgaris. Molecular General Genetics, 198, 42-49.
[8] Anthony, J.L., VonderHaar, R.A. and Hall, T.C. (1990) Nucleotide Sequence of an α-Phaseolin Gene from Phaseolus vulgaris. Nucleic Acid Research, 18, 11.
[9] Sun, S.M., Mcleester, R.C., Bliss, F.A. and Hall, T.C. (1974) Reversible and Irreversible Dissociation of Globulins from Phaseolus vulgaris Seed. Journal of Biological Chemistry, 249, 2118-2121.
[10] Sun, S.M., Slightom, J.L. and Hall, T.C. (1981) Intervening Sequences in a Plant Gene-Comparison of the Partial Sequence of cDNA and Genomic DNA of French Bean Phaseolin. Nature, 289, 37-41.
[11] Slightom, J.L., Sun, S.M. and Hall, T.C. (1983) Complete Nucleotide Sequence of a French Bean Storage Protein Gene: Phaseolin. Proceedings of the National Academy of Sciences of the United States of America, 80, 1897-1901.
[12] Bustos, M.M., Begum, D., Kalkan, F.A., Battrawand, M.J. and Hall, T.C. (1991) Positive and Negative cis-Acting DNA Domains Are Required for Spatial and Temporal Regulation of Gene Expression by a Seed Storage Protein Promoter. European Molecular Biology Organization Journal, 10, 1469-1479.
[13] Burow, M.D., Sen, P., Chlan, C.A. and Murai, N. (1992) Developmental Control of the β-Phaseolin Gene Requires Positive, Negative, and Temporal Seed-Specific Transcriptional Regulatory Elements and a Negative Element for Stem and Root Expression. Plant Journal, 2, 537-548.
[14] Hoffman, L.M., Donaldson, D.D. and Herman, E.M. (1988) A Modified Storage Protein Is Synthesized, Processed, and Degraded in the Seeds of Transgenic Plants. Plant Molecular Biology, 11, 717-729.
[15] Lawrence, M.C., Izard, T., Beuchat, M., Blagroveand, R.J. and Colman, P.M. (1994) Structure of Phaseolin at 2·2 Å Resolution: Implications for a Common Vicilin/Legumin Structure and the Genetic Engineering of Seed Storage Proteins. Journal of Molecular Biology, 238, 748-776.
[16] Lawrence, M.C., Suzuki, E., Varghese, J.N., Davis, P.C., Van Donkelaar, A., Tulloch, P.A. and Colman, P.M. (1990) The Three-Dimensional Structure of the Seed Storage Protein Phaseolin at 3 Å Resolution. European Molecular Biology Organization Journal, 9, 9-15.
[17] Dyer, J.M., Nelsonand, J.W. and Murai, N. (1992) Biophysical Analysis of Phaseolin Denaturation Induced by Urea, Guanidinium Chloride, pH, and Temperature. Journal of Protein Chemistry, 11, 281-288.
[18] Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Halland, T.C. and Kemp, J.D. (1985) Developmentally Regulated Expression of the Bean β-Phaseolin Gene in Tobacco Seed. Proceedings of the National Academy of Sciences of the United States of America, 82, 3320-3324.
[19] Greenwood, J.S. and Chrispeels, M.J. (1985) Correct Targeting of the Bean Storage Protein Phaseolin in the Seeds of Transformed Tobacco. Plant Physiology, 79, 65-71.
[20] Barker, S.J., Harada, J.J. and Goldberg, R.B. (1988) Cellular Localization of Soybean Storage Protein mRNA in Transformed Tobacco Seeds. Proceedings of the National Academy of Sciences of the United States of America, 85, 458-462.
[21] Marcellino, L.H., Neshich, G., Grossi de Sá, M.F., Krebbers, E. and Gander, E.S. (1996) Modified 2S Albumins with Improved Tryptophan Content Are Correctly Expressed in Transgenic Tobacco Plants. Federation of European Biochemical Societies Letter, 385, 154-158.
[22] Burow, M.D., Chlan, C.A., Sen, P., Lisca, A. and Murai, N. (1990) High-Frequency Generation of Transgenic Tobacco Plants after Modified Leaf Disk Cocultivation with Agrobacterium tumefaciens. Plant Molecular Biology Reporter, 8, 124-139.
[23] Sen, P., Chlan, C.A., Burow, M.D., Lee, W.S. and Murai, N. (1993) Apical and Lateral Shoot Apex-Specific Expression Is Conferred by Promoter of the Seed Storage Protein β-Phaseolin Gene. Transgenic Research, 2, 21-28.
[24] Sambrook, D., Fritsh, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory.
[25] Wang, H., Meiqing, Q.L. and Cutler, A.J. (1993) A Simple Method of Preparing Plant Samples for PCR. Nucleic Acids Research, 21, 4153-4154.
[26] Bollini, R. and Chrispeels, M.J. (1979) The Rough Endoplasmic Reticulum Is the Site of Reserve Protein Synthesis in Developing Phaseolus vulgaris Cotyledons. Planta, 146, 487-501.
[27] Bollini, R., van der Wilden, W. and Chrispeels, M.J. (1982) A Precursor of the Reserve-Protein, Phaseolin, Is Transiently Associated with the Endoplasmic Reticulum of Developing Phaseolus vulgaris Cotyledons. Physiologia Plantarum, 55, 82-92.
[28] Bollini, R., Vitale, A. and Chrispeels, M.J. (1983) In Vivo and in Vitro Processing of Seed Reserve Protein in the Endoplasmic Reticulum: Evidence for Two Glycosylation Steps. Journal of Cell Biology, 96, 999-1007.
[29] D’Amico, L., Valsasina, B., Daminati, M.G., Fabbrini, M.S., Nitti, G., Bollini, R., Ceriotti, A. and Vitale, A. (1992) Bean Homologs of the Mammalian Glucose-Regulated Proteins: Induction by Tunicamycin and Interaction with Newly Synthesized Seed Storage Proteins in the Endoplasmic Reticulum. Plant Journal, 2, 443-455.
[30] Ceriotti, A., Pedrazzini, E., Bielli, E., Giovinazzo, A., Bollini, R. and Vitale, A. (1995) Assembly and Intracellular Transport of Phaseolin, the Major Storage Protein of Phaseolus vulgaris L. Journal of Plant Physiology, 145, 648-653.
[31] Lupattelli, F., Pedrazzini, E., Bollini, R., Vitale, A. and Ceriotti, A. (1997) The Rate of Phaseolin Assembly Is Controlled by the Glucosylation State of Its N-Linked Oligosaccharide Chains. Plant Cell, 9, 597-609.
[32] Chrispeels, M.J. (1991) Sorting of Proteins in the Secretory System. Annual Review of Plant Physiology and Plant Molecular Biology, 42, 21-53.
[33] Frigerio, L., de Virgillio, M., Prada, A., Faoro, F. and Vitale, A. (1998) Sorting of Phaseolin to the Vacuole Is Saturable and Requires a Short C-Terminal Peptide. Plant Cell, 10, 1031-1042.
[34] Liu, J.-X. and Howell, S.H. (2010) Endoplasmic Reticulum Protein Quality and Its Relationship to Environmental Stress Responses in Plants. Plant Cell, 22, 2930-2942.
[35] Xu, C., Wang, S., Thibault, G. and Ng, D.T.W. (2013) Futile Protein Folding Cycles in the ER Are Terminated by the Unfolded Protein O-Mannosylation Pathway. Science, 340, 978-981.
[36] Pedrazzini, E., Giovinazzo, G., Bollini, R., Ceriotti, A. and Vitale, A. (1994) Binding of BiP to an Assembly-Defective Protein in Plant Cells. Plant Journal, 5, 103-110.
[37] Vitale, A., Bielli, A. and Ceriotti, A. (1995) The Binding Protein Associates with Monomeric Phaseolin. Plant Physiology, 107, 1411-1418.
[38] Smith, M.H., Ploegh, H.L. and Weissman, J.S. (2011) Road to Ruin: Targeting Proteins for Degradation in the Endoplasmic Reticulum. Science, 334, 1086-1090.
[39] Sturm, A., Van Kuk, J.A., Vliegenthart, J.F.G. and Chrispeels, M.J. (1987) Structure, Position, and Biosynthesis of the High Mannose and the Complex Oligosaccharide Side-Chains of the Bean Storage Protein Phaseolin. Journal of Biological Chemistry, 262, 13392-13403.
[40] Sturm, A., Johnson, K.D., Szumilo, T., Elbein, A.D. and Chrispeels, M.J. (1987) Subcellular Localization of Glycosidases and Glycosyltransferases Involved in the Processing of N-Linked Oligosaccharides. Plant Physiology, 85, 741-746.
[41] Bustos, M.M., Kalkan, F.A., Vanden Bosch, K.A. and Hall, T.C. (1991) Differential Accumulation of Four Phaseolin Glycoforms in Transgenic Tobacco. Plant Molecular Biology, 16, 381-395.
[42] Harada, T., Miyairi, K. and Murai, N. (2010) Trimeric Glycoproteins of Bean Seed Storage Protein Phaseolin Were Purified from Baculovirus-Infected Insect Sf9 Cells for Use of Structural Study. Plant Science, 179, 123-132.
[43] Pueyo, J.J., Chrispeels, M.J. and Herman, E.M. (1995) Degradation of Transport-Competent Destabilized Phaseolin with a Signal for Retention in the Endoplasmic Reticulum Occurs in the Vacuole. Planta, 196, 586-596.
[44] Figerio, L., Pastres, A., Prada, A. and Vitale, A. (2001) Influence of KDEL on the Fate of Trimeric or Assembly-Defective Phaseolin: Selective Use of an Alternative Route to Vacuoles. Plant Cell, 13, 1109-1126.
[45] Foresti, O., Frigerio, L., Holkeri, H., de Virgilio, M., Vavassori, S. and Vitale, A. (2003) A Phaseolin Domain Involved Directly in Trimer Assembly Is a Determinant for Binding of the Chaperone BiP. Plant Cell, 15, 2464-2475.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.