Regulation of the Activity and Location of Starch-Related Dikinases

少量淀粉相关葡糖基残基的可逆磷酸化由两种质体二激酶（葡聚糖，水二激酶[GWD]和磷酸葡聚糖，水二激酶[PWD]）和至少两种磷酸酶（SEX 4和LSF 2）的协同作用介导。可逆的葡聚糖改性对于正常的淀粉周转和植物生长是必不可少的。该提议使用各种实验方法并集中在以下方面：a）GWD催化意想不到的大量磷酸转移反应。先前的结果已经表明，至少在γ-磷酸从ATP转移到例如水的过程中，氨基酸残基被瞬时磷酸化。使用各种截短和突变的GWD版本和质谱法，将鉴定这种瞬时磷酸化的氨基酸。比较PWD的磷酸转移。B）分析二激酶所使用的葡聚糖性质。因此，将使用各种淀粉和新直链淀粉级分并将其表征为二激酶的底物。此外，还将在体外和体内分析GWD和PWD在淀粉上潜在结合位点的分布。随着越来越多的证据表明GWD参与淀粉合成，GWD对淀粉合成和淀粉颗粒形态形成的贡献将被分析。因此，在不同的光条件下，诱导和部分补充GWD转基因植物的淀粉颗粒形态和表面特性进行了研究。

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.