Gene, Enzyme and Physiological Characterization of Shrunken-2 Isoalleles

点击翻译按钮获取中文摘要

Starch comprises one of the major glucose polymers in all living organisms. It is essential for plant life since it provides a mechanism to store the vast amounts of fixed carbon and, as the the major component of many important seeds, starch provides the important energy sourcetransferred from parent to progeny. Animal life is also dependent on starch since animals (including humans) rely primarily on the starch-rich cereal seeds for essential calories and nutrients. Because of the importance of starch to both plant and animal life, the Hannah lab hasfocused on the enhancement of starch content in cereal seeds through genetic manipulation of the rate-limiting enzyme, ADP-glucose pyrophosphorylase (AGP). AGP catalyzes the synthesis of ADP-glucose and pyrophosphate from glucose-1-PO4 and ATP. ADP-glucose then serves as the glucose donor for the carbon found in starch. The work to be performed focuses on the allosteric properties of AGP. The triose sugar, 3-phosphoglyceric acid (3-PGA) activates and phosphate inhibits AGP. Specific aims include determination of whether the low level of 3-PGA activation of the maize endosperm AGP reflects loss of activation potential or an independence of 3-PGA for maximum activity. Other experiments include analysis of hybrid AGPs involving the potato tuber small subunit and the maize large subunit to identify sites involved in allosteric regulation and catalysis as well as determination of whether the strikingly different levels of activity seen in maize and in E. coli of an allosterically-altered maize AGP variant termed Sh2-Rev6 involves differential modification of the inserted amino acids. Amino acid insertions at this position that do not restore activity will be identified. Finally, a large scale mutagenesis project will be initiated to identify motifs involved in enzyme assembly, catalysis and allosteric regulation. The impressive field performance of Sh2-Rev6 in maize and now in another monocot as well as in a dicot points up the critical importance of the regulatory properties of AGP. The studies to be performed here should provide definitive insight into the mechanism of Sh2-Rev6 action as well as possibly identify other important variants.