Detailed analyses of starch granule surface structure alterations

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Starch degradation is controlled by a phosphorylation/dephosphorylation cycle in which first the surface of the starch granules is phosphosphorylated by α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). This decreases the surface crystallinity and transforms the highly ordered glucan chains of amylopectin into a solubilized state. These solubilized glucan chains provide suitable substrates for hydrolases such as β-amylases and isoamylases, with the orchestrated action of the phosphatases SEX4 and LSF2 being obligatory. To date, little is known about the regulation of these processes at the starch granule surface. At the same time, it also remains unclear in detail how the surface structures/properties influence the enzymes involved in starch metabolism and, vice versa, how these surface properties/morphologies of the granules are altered in detail by the action of specific proteins/enzymes. For the recently identified protein ESV1, which is essential for the rate of starch degradation during the dark phase, it was shown in in vitro studies that it alters dikinase-mediated phosphate incorporation. Similarly, this effect occurred with the homolog LESV. It was found that these effects were not caused by protein-protein interactions, but rather by the binding of ESV1 and LESV at specific structures at the starch granule surface. It was shown that the two proteins differ in their glucan binding specificity, with ESV1 preferentially binding to amylopectin and LESV to amylose. An influence of ESV1 and LESV on other enzymes involved in starch metabolism such as BAM, ISA, SEX4, PHS1, and SS1 was also confirmed. However, the exact molecular background is unclear. In this proposal, this mechanism will be analysed in detail using different experimental approaches and considering the following main objectives. (1) Identifying the glucan structures bound by proteins ESV1 and LESV and which protein region is responsible for the observed effects on enzymes involved in starch metabolism. (2) The analysis of other enzymes/proteins involved in starch metabolism that are affected by ESV1 and LESV. (3) Investigating the phosphorylation pattern of GWD and PWD from different species and the influence of ESV1 and LESV on this pattern. (4) The analysis of the surface structural characteristics of starch granules in different mutants during the diurnal cycle and aging process, and how these structural properties determine granule characteristics as size, size distribution and morphology.