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

Abstract

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.