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Regulation of starch biosynthesis in Arabidopsis thalinan

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Title: Regulation of starch biosynthesis in Arabidopsis thalinan
Author: Zhao, Qianru
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Emes, Michael
Abstract: Starch is an important carbohydrate in higher plants and is widely used in food and non-food industries. Starch is synthesized in plastids through the actions of starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (DBE), each with multiple isoforms. This study tested the hypothesis that SSs and SBEs in Arabidopsis thaliana (Arabidopsis) chloroplasts, involved in transient starch biosynthesis in leaves, are regulated by protein phosphorylation and protein-protein interactions. The sub-organelle distribution of starch biosynthetic enzymes was determined and SS2 and SBE2.2 were detected in wild type starch granules at the end of a 16/8h light/dark photoperiod. However, starch granules of ss2- and ss3- mutants also lacked SBE2.2, suggesting an interaction between SS2 and SBE2.2, and also with SS3 in the chloroplast stroma. Formation of protein complexes between SS1, SS2 and SBE was investigated by studying starch granule bound proteins in sbe- mutants and co-immunoprecipitation of interacting enzymes. In wild-type Arabidopsis chloroplasts, SS1 co-immunoprecipitated with SBE2.1 and SBE2.2, and was previously observed in cereal endosperm, suggesting that the interaction mechanisms among starch biosynthetic enzymes are common across species. Recombinant SBE2.1 and SBE2.2 were shown to be phosphorylated by chloroplast protein kinases. Two phosphoserine residues that are highly conserved within the SBEII class of enzyme were identified by site-directed mutagenesis. A phosphorylation-dependent protein complex could be formed in vitro between recombinant Arabidopsis SBE and maize SSI and SSII. However, mutation of the identified SBE phosphorylation sites did not lead to disruption of interactions with SSIIa or SSI, suggesting that these sites are not directly linked to the formation of such protein complexes. When SBE2.2, D653 was mutated to Ser, ATP-dependent protein-protein interaction between maize SSI, SSIIa and Arabidopsis SBE2.2 were lost, suggesting this residue is important in regulating interactions with the SSI and SSII classes of starch synthases. The results of this study provide new insight into the regulation of transient starch biosynthesis in Arabidopsis chloroplasts.
URI: http://hdl.handle.net/10214/8787
Date: 2015-04


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