Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/1997
Publication Date: N/A
Citation: N/A Interpretive Summary: Because soybean is an important agronomic crop in the United States, research is carried out by USDA-ARS to improve seed quality. A persistent problem has been that soybean proteins have an inferior nutritional quality due to suboptimal amounts of essential sulfur amino acids. Despite extensive research, conventional plant breeding approaches have been unable eto improve soybean nutritional quality and attention has now focused on biotechnological approaches. Successful application of a biotechnological approach requires detailed knowledge about the structure of the molecules targeted for modification and the mechanisms involved in the synthesis and assembly of those molecules. To facilitate the study of glycinin, the most prevalent protein in most soybeans, a procedure has been developed to synthesize glycinin subunits in a cell free system and assemble them into subunit complexes like those found in the seed. The research outlined in this paper show that the two cysteine amino acids involved in holding together the two chains of the mature glycinin together via a disulfide bridge perform a critical function during subunit folding. Not only are they required for optimal folding and assembly of glycinin trimers, but the disulfide bridge is necessary for glycinin trimers to assemble into hexamers. The information in this paper provides insight about the pathway followed during the maturation of glycinin storage proteins and will be useful to scientists working to improve the quality of grain legumes.
Technical Abstract: Seed 11S globulins contain two conserved disulfide bonds, one connecting the acidic and basic chains of the mature subunit (inter-chain disulfide bond), and a second connecting parts of the acidic chain (intra-chain disulfide bond). To investigate the role of these disulfide bonds in subunit assembly and post-translational processing, cDNAs were constructed that encoded mutant Vicia faba legumin B subunits with disrupted intra- and interchain disulfide bonds. When expressed in vitro, assembly of unmodified proglobulin trimers was stimulated kinetically by the addition of oxidized glutathione. This stimulation was not detected in assembly of mutant subunits without the inter-chain disulfide bond. When unmodified prolegumins were cleaved into acidic and basic chains in vitro, an oxidizing reaction condition was required for formation of hexamers. Subunits lacking the interchain disulfide bond were incapable of assembly into hexamers despite being correctly cleaved into acidic and basic polypeptides. The cDNA constructs encoding the unmodified and mutant prolegumin subunits were transformed into tobacco. Acidic and basic chains of both mutant and unmodified subunits were found to accumulate in protein bodies of transgenic tobacco seeds as hexamers. Assembly in seeds of transgenic tobacco probably involved formation of mixed complexes composed of endogenous tobacco 11S globulins and the mutant legumin B subunits. To test this possibility, mixed trimers composed of both mutant and unmodified subunits were assembled in vitro. Unlike trimers composed of only mutant subunits, trimers made from mixtures of mutant and unmodified subunits were capable of hexamer assembly. These results identify the disulfide bonds as important factors that influence the assembly of 11S globulins.