Loss of the granule-bound starch synthase protein reshapes the starch proteome and phosphorylation profile in potato tuber

Amylose and amylopectin define the structural behavior, gelatinization and retrogradation of starch, while starch phosphorylation modulates its hydration, viscosity, and stability. Granule-bound starch synthase (GBSS) is responsible for amylose biosynthesis, and phosphorylation is catalyzed by the granule associated glucan- and phosphoglucan- water dikinases, (GWD and PWD), respectively. To explore how GBSS influences the association of other starch-binding proteins, we analyzed the starch proteome of two edited tetraploid potato lines using clustered regularly interspaced short palindromic repeats associated with protein 9 technology (CRISPR/Cas9): one lacking GBSS entirely, and one retaining 16% residual expression. Despite reduced total protein content in both lines (by 50% and 35%, respectively), most starch-bound enzymes increased in abundance by 3 to 5-fold, except protein targeting to starch 1 (PTST1), which was absent. This suggests that GBSS loss promotes recruitment of alternative starch-metabolizing enzymes. Elevated levels of granule-bound GWD in the mutants correlated with increased level of C6 phosphate by 1.42-fold, linking GBSS disruption to changes in starch phosphorylation. Moreover, the absence of PTST1 in both lines further supports its role in targeting GBSS to starch granules, providing further insight into the protein-protein interactions governing starch biosynthesis in potato. Overall, the loss of GBSS reorganizes the starch-bound proteome, with most of the other protein quantity being increased, including the dikinases, consequently increasing the granule phosphorylation. These findings highlight how GBSS deficiency reshapes starch composition and proteome organization, with implications for starch structure and functionality.