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Post-Translational Regulation of Starch Branching Enzyme 2.2 from Arabidopsis thaliana

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Title: Post-Translational Regulation of Starch Branching Enzyme 2.2 from Arabidopsis thaliana
Author: MacNeill, Gregory
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Emes, MichaelTetlow, Ian
Abstract: Starch is the most abundant carbohydrate storage molecule in plants. It is produced in chloroplasts in the light providing a source of fixed carbon when photosynthesis is not occurring. Its synthesis involves the coordinated activity of multiple enzyme classes, including starch branching enzymes (SBE) which introduce branch points onto linear glucan chains. Two functional SBE isoforms (SBE2.1 and SBE2.2) exist in Arabidopsis, with SBE2.2 accounting for most of the branching activity. Loss of both isoforms results in the abolition of starch production, with fixed carbon accumulating in high levels of maltose/maltodextrins. Previous studies have identified the role of protein phosphorylation in formation of heteromeric enzyme complexes between SBEs and starch synthases (SS). Here, a combination of bioinformatics, in vitro analysis, and in vivo complementation approaches were used to identify sites of post-translational regulation of AtSBE2.2 and analyse their importance. Two previously identified phosphorylation sites were investigated for their role in catalytic activity and protein-protein interactions. Phosphorylation of Ser290 was shown to be involved in interactions with SS2, while phosphorylation of Ser301 may affect catalytic activity by stabilizing a glucan binding site through interaction with a conserved domain (KCRRR). Redox regulation of recombinant SBE2.2 was investigated and three cysteines critical for enzyme activity (Cys375, Cys515 and Cys744) were identified. Confounding factors in methodology commonly used to modify redox state and investigate SBE activity were identified, casting doubt on previous reports. Site directed mutagenesis of phosphorylation sites, the KCRRR domain and Cys744 was performed and the products were expressed in either a wildtype or an SBE-null Arabidopsis genotype. Starch production and growth were partially restored by complementation of the SBE-null genotype with the WT SBE2.2 protein, as well as by some of the mutated sequences. The results support the hypothesis that phosphorylation of Ser290 and Ser301 play distinct roles, governing protein interactions and glucan binding, respectively. Mutation of Cys744 had no apparent effect on growth or starch production. Expression of SBE2.2 with a mutation to the KCRRR domain in the Landsberg erecta genotype resulted in unique floral and silique morphology, as well as increased seed production, with potential applications for crop production.
Date: 2020-10
Rights: Attribution-NonCommercial-ShareAlike 4.0 International
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Attribution-NonCommercial-ShareAlike 4.0 International Except where otherwise noted, this item's license is described as Attribution-NonCommercial-ShareAlike 4.0 International