Regulation of the Activity and Location of Starch-Related Dikinases

少量淀粉相关葡萄糖基残基的可逆磷酸化是由两种质体二激酶（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（SEX4和LSF2）协同作用介导的。可逆的葡聚糖修饰对于正常的淀粉周转和植物生长是必不可少的。本文采用多种实验方法，重点研究了以下几个方面：1)GWD催化的磷转移反应出乎意料地多。先前的结果表明，至少在从ATP转移到水的过程中，氨基酸残基会短暂磷酸化。使用各种截断和突变的GWD版本和质谱，将鉴定这种瞬时磷酸化的氨基酸。对比分析了PWD的磷光转移。b)分析二激酶所使用的葡聚糖性质。因此，各种淀粉和新直链淀粉馏分将被用作二激酶的底物。此外，我们将在体内和体外分析GWD和PWD在淀粉上的潜在结合位点分布。c)随着越来越多的证据表明GWD参与淀粉合成，我们将分析GWD对淀粉合成和淀粉颗粒形态形成的贡献。因此，我们将在不同光照条件下，对诱导型和部分补型GWD转基因植株的淀粉颗粒形态和表面特性进行研究。

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.