Studies on modulation of the expression of Gy2 glycinin gene and on assembly properties of modified beta-conglycinin subunits

The participation of regulatory elements in the expression of seed storage protein genes is not based on a demonstration of function. This work was to verify whether the regulatory functions attributed to 5$\sp\prime$-CATGCAT-3$\sp\prime$ elements, the legumin box, and the 10 base pair inverted repeats could be demonstrated in vivo. The promoter of the Gy2 soybean glycinin gene received several independent alterations. Plant transformation indicates that alterations on or around the proximal 5$\sp\prime$-CATGCAT-3$\sp\prime$ element that lies within the legumin box results in significative reduction in $\beta$-glucuronidase activity accumulated in seeds. Either elimination of the distal 5$\sp\prime$-CATGCAT-3$\sp\prime$ element or base pair replacements of the distal inverted repeat did not cause significant reduction in the activity. Alteration of the proximal inverted repeat decreased the activity in tobacco seeds, while no significant decrease was observed in the activity found in Arabidopsis seeds. The amount of activity is approximately threefold greater when the 2.3 kb promoter is used instead of the 0.4 kb promoter. No activity was found in organs other than seeds. The major seed storage proteins of soybeans, 11S (glycinin) and 7S ($\beta$-conglycinin), are co-localized in the same storage vacuoles. Interaction among oligomers of these proteins has not been demonstrated. These proteins share a number of predicted secondary structures. The 11S Asn-Gly proteolytic cleavage site is preserved evolutionarily among the homologous 11S family, but it is naturally absent in 7S proteins. This work shows that mutant subunits of $\beta$-conglycinin containing the Asn-Gly cleavage site self-assemble into trimers in vitro and do not differ from the unmodified ones when compared in their rate of assembly. Unlike the wild-type 7S subunits, the mutant trimers are processed when exposed in vitro to the protease responsible for proteolytic cleavage of the 11S proteins. After separation of the cleaved subunits on a sucrose gradient, only trimers are found. The processed 7S trimers did not interact with native glycinin subunits during reassociation