Investigation of the function of branching enzymes I, IIa, and IIb in the determination of amylopectin structure and regulation of starch biosynthesis

Marsh, Rebekah
Major Professor
Alan M. Myers
Martha G. James
Committee Member
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Biochemistry, Biophysics and Molecular Biology

Starch is one of the most abundant carbohydrates on earth serving vital roles as the primary energy reserve of plants, a significant source of calories in the human diet, and a renewable resource for other industrial applications. The multiplicity of starch biosynthetic enzymes is evolutionarily conserved in plants suggesting their functions are essential in starch biosynthesis. The overall goal of this research is to determine the specific molecular functions of the multiple starch branching enzyme (BE) isoforms in maize.;This dissertation first addresses what, if any, are the differences in enzymatic properties of BEIIa and BEIIb. Two polyclonal antisera were produced, alphaBEI and alphaBEIIab which specifically identify BEI and both BEII isoforms, respectively. Chromatographic purification techniques separated native BEIIa or BEIIb from the other two branching enzymes. The capacities to modify the structure of amylose were compared between native BEIIa and BEIIb and recombinant BEIIa (rIIa). The evolutionary conservation of BEIIa and BEIIb is not accounted for by their enzymatic differences. Differences in the glucan chain length distribution created by native BEIIa and rIIa suggest that in vivo there are factors affecting the activity of BEs.;This dissertation next addresses are branching enzymes involved in multi-subunit complexes (MSCs)? This research provides direct evidence BEIIa and SSI interact with each other in MSC and that SSIII may be a component of the MSCs. Indirect evidence from GPC analysis of BEIIb and multiple electrophoretic mobility forms of BEI suggests that they may be involved in MSCs. Pleiotropic effects in branching enzyme mutants on starch synthase (SS) activity revealed at least three SS band activities are affected when BEIIa was mutated. Further research is needed to investigate the functional significance of the complexes involving BEIIa, SSI and possibly SSIII.;This dissertation addresses what branching enzyme isoforms exist in leaves as compared to endosperm. The same BEI polypeptide present in maize endosperm was found to be present in maize leaves. Leaf BEI was found to be similar to endosperm BEI in predicted molecular weight, approximately 80 kDa, in molecule size, a monomeric protein, and also possesses multiple electrophoretic mobility forms.