| Starch is widely used in food and non-food sectors. Its biosynthesis is known to involve at least four groups of committed enzymes: ADP-glucose pyrophosphorylase, starch synthases (SS), starch branching enzymes (SBE), and debranching enzymes (DBE). Plastidial starch phosphorylase (SP) is, also suggested to be involved in starch biosynthesis. Significant proportions of the starch biosynthetic enzymes, particularly SSI, SSIIa, and SBEIIb are bound to starch granules in maize. In this thesis, biochemical studies on protein-protein interactions between key enzymes of the starch biosynthetic pathway in wheat and maize endosperms, identify a potentially important mechanism of regulating starch biosynthesis by formation of phosphorylation-dependent multi-enzyme complexes between isoforms of starch synthases and branching enzymes. Furthermore, studies on a well-characterized maize mutant lacking the dominant branching enzyme activity, SBEIIb, amylose extender (ae-), demonstrated distinct patterns of protein-protein interactions compared with wild-type, suggesting functional complementation for the loss of SBEIIb in protein complexes by SBEI, SBEIIa and SP. Co-immunoprecipitation using peptide-specific antibodies showed that in amyloplasts from normal maize endosperm, protein-protein interactions involving SSI, SSIIa, and SBEIIb could be detected. By contrast, in ae- amyloplasts, SSI and SSIIa were shown to interact with SBEI, SBEIIa and SP. All interactions in normal maize were strongly enhanced by ATP, and reduced by the addition of alkaline phosphatase, indicating a role for protein phosphorylation in assembly. All the protein-protein interactions observed in wild-type and the ae- mutant occur during the grain-filling stage of endosperm development. Interactions were also reconstituted in vitro using recombinant forms of SSs and SBEs. This study proposes that during. amylopectin biosynthesis in maize amyloplasts, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in wild-type endosperm, recruits SBEIIb, but when SBEIIb is absent (ae-), recruits SBEI, SBEIIa and SP. These differences in protein complexes are mirrored in the complement of starch synthesizing enzymes detected in starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis. |
