Regulation of the Activity and Location of Starch-Related Dikinases

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The reversible phosphorylation of a low amount of starch-related glucosyl residues is mediated by a concerted action of two plastidial dikinases (glucan, water dikinase [GWD] and phosphoglucan, water dikinase [PWD]) and, at least, two phosphatases (SEX4 and LSF2). The reversible glucan modification is essential for normal starch turn-over and for plant growth. This proposal uses various experimental approaches and focuses on the following aspects: a) GWD catalysis an unexpected high number of phosphotransfer reactions. Previous results have shown that, at least, during the transfer of the gamma-phosphate from ATP to e.g. water an amino acid residue is transiently phosphorylated. Using various truncated and mutated GWD versions and mass spectrometry, this transiently phosphorylated amino acid will be identified. In comparison the phosphotransfer of PWD will be analyzed. b) The glucan properties used by the dikinases will be analyzed. Therefore, various starch and neoamylose fractions will be used and characterized as substrate for the dikinases. In addition the distribution of potential binding sites of GWD and PWD on starches will be analyzed in vitro and in vivo. c) As increasing evidences exist, that GWD is involved in starch synthesis, the contribution of GWD to starch synthesis and formation of starch granule morphology will be analyzed. Therefore, starch granule morphology and surface properties will be investigated in inducible and partially complemented GWD transgenic plants under various light conditions.