Detailed analyses of starch granule surface structure alterations

淀粉降解受磷酸化/去磷酸化循环控制，在这个循环中，淀粉颗粒的表面首先被α-葡聚糖、水二激酶（GWD）和磷酸葡聚糖水二激酶（PWD）磷酸化。这降低了表面结晶度，并将支链淀粉的高度有序的葡聚糖链转化为可溶解状态。这些溶解的葡聚糖链为水解酶（如β-淀粉酶和异淀粉酶）提供了合适的底物，磷酸酶SEX4和LSF2的协同作用是必需的。迄今为止，对淀粉颗粒表面这些过程的调控知之甚少。同时，表面结构/性质如何影响淀粉代谢所涉及的酶，反之亦然，颗粒的这些表面性质/形态如何通过特定蛋白质/酶的作用而改变，这些细节也尚不清楚。对于最近发现的蛋白质ESV1，它对淀粉在黑暗阶段的降解速率至关重要，体外研究表明，它改变二激酶介导的磷酸结合。类似地，这种效应也发生在同源的LESV上。研究发现，这些作用不是由蛋白-蛋白相互作用引起的，而是由ESV1和LESV在淀粉颗粒表面特定结构上的结合引起的。结果表明，这两种蛋白的葡聚糖结合特异性不同，ESV1优先与支链淀粉结合，LESV优先与直链淀粉结合。ESV1和LESV对其他参与淀粉代谢的酶如BAM、ISA、SEX4、PHS1和SS1也有影响。然而，确切的分子背景尚不清楚。在本提案中，将使用不同的实验方法详细分析该机制，并考虑以下主要目标。(1)确定了ESV1和LESV蛋白结合的葡聚糖结构，以及哪个蛋白区域负责观察到的对淀粉代谢相关酶的影响。(2)其他受ESV1和LESV影响的淀粉代谢相关酶/蛋白分析。(3)研究不同物种GWD和PWD的磷酸化模式，以及ESV1和LESV对这一模式的影响。(4)分析不同突变体淀粉颗粒在日循环和老化过程中的表面结构特征，以及这些结构特性如何决定颗粒的大小、粒度分布和形态特征。

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.