Skip to main content
ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Plant Physiology and Genetics Research » Research » Publications at this Location » Publication #316860

Research Project: Molecular Genetic Analysis of Abiotic Stress Tolerance and Oil Production Pathways in Cotton, Bioenergy and Other Industrial Crops

Location: Plant Physiology and Genetics Research

Title: Modified bean seed protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations

Author
item LASSERRE, ERIC - Louisiana State University
item KO, T - Louisiana State University
item Dyer, John
item MURAI, NORIMOTO - Louisiana State University

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2015
Publication Date: 3/17/2015
Publication URL: http://handle.nal.usda.gov/10113/61034
Citation: Lasserre, E., Ko, T.S., Dyer, J.M., 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.

Interpretive Summary: Legumes and cereals are major sources of protein for humans and domesticated animals, but their nutritional value is somewhat limited due to a low content of essential amino acids. For instance, legumes are typically deficient in the essential amino acid methionine, while cereals are deficient in lysine and threonine. Although breeding efforts have been used successfully to improve the nutritional content of maize, efforts to increase the methionine content of legumes remains a challenge. As an alternative approach to increase methionine content of legumes, scientists at Louisiana State University, the Universite de Perpignan in France, and the ARS lab in Maricopa, AZ, determined whether the major storage protein of common beans could be engineered to contain higher amounts of methionine, while preserving the ability of the protein to accumulate to high levels in plant seeds. Towards this end, a variety of modified proteins were tested, with varying amounts of additional methionine residues, and the proteins were subsequently expressed in either bacterial cells or in transgenic plants. The results revealed that while the proteins could indeed be produced in bacteria, the proteins did not accumulate in plant seeds. These data suggest that plants contain a stringent quality control mechanism that regulates the types and amounts of protein that accumulate in developing seeds. These findings, while negative in nature, will be particularly important for scientists interested in exploring various avenues for improving nutritional content of legumes, which serve as a major source of protein for the developing world.

Technical 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 beta-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 beta-sheets and tum/loop regions, respectively, of N- and C-barrel structures.