Post-translational regulation of starch synthesis in higher plants

本提案的主要目的是提高我们对植物淀粉合成调控的理解，并确定有助于淀粉结构组织的生化机制。淀粉是一种天然存在的葡萄糖聚合物，广泛用于食品和非食品工业。功能的变化源于直链淀粉与支链淀粉的比例、葡聚糖链长、分支和颗粒大小等性状。这两个pi是第一个证明淀粉合成酶聚集形成异质蛋白复合物，由可逆的蛋白质磷酸化催化。最近，我们利用遗传方法证明，淀粉合成酶IIa和淀粉分支酶IIb的突变不仅会影响酶活性的丧失，还会改变异质蛋白复合物的性质，从而影响淀粉颗粒的大小、结构和性质。我们认为蛋白质磷酸化是决定淀粉合成酶之间物理联系的关键，影响所产生的葡聚糖聚合物的结构。为了验证这一假设，我们将：a)确定相关蛋白之间的相互作用域；B)确定体内磷酸化位点和每种酶磷酸化状态被修饰的条件；C)表征负责淀粉合成酶翻译后修饰的蛋白激酶和磷酸酶；D)在体内突变淀粉合成酶的磷酸化位点和蛋白质相互作用域，并表征由此产生的淀粉表型的结构。这些研究将使我们能够为翻译后修饰在淀粉合成和结构调节中的作用建立一个模型。该计划代表了两个pi的自然协同作用，将植物生理学，细胞生物学，生物化学，遗传学，淀粉和蛋白质化学结合在一起，研究植物代谢最重要的途径之一。所产生的知识有可能应用于为食品和工业部门的特定应用量身定制淀粉。

The primary objective of this proposal is to enhance our understanding of the regulation of starch synthesis in plants and identify the biochemical mechanisms which contribute to the organisation of starch structure. Starch is a naturally occurring glucose polymer and is used widely in food and non-food industries. Variation in functionality is derived from traits such as the ratio of amylose to amylopectin, glucan chain length, branching and granule size. The two PIs were the first to demonstrate that enzymes of starch synthesis aggregate to form heteromeric protein complexes, catalysed by reversible protein-phosphorylation. Recently we have made use of genetic approaches to demonstrate that mutations in starch synthase IIa and starch branching enzyme IIb have an impact beyond the loss of enzyme activity, modifying the nature of the heteromeric protein-complexes with consequences for the size, structure and properties of the starch granule. We propose that protein phosphorylation is critical in determining physical association among enzymes of starch synthesis, affecting the structure of the glucan polymer produced. To test this hypothesis we will: a) determine the interaction domains between associated proteins; b) identify phosphorylation sites and conditions in vivo under which the phosphorylation state of each enzyme is modified; c) characterise the protein kinase(s) and phosphatase(s) responsible for post-translational modification of enzymes of starch synthesis; d) mutate the phosphorylation sites and protein-interaction domains of starch synthesising enzymes in vivo and characterise the structure of the resulting starch phenotypes. These studies will allow us to develop a model for the role of post-translational modification in the regulation of starch synthesis and structure. The programme represents a natural synergy of the two PIs, bringing together plant physiology, cell biology, biochemistry, genetics, and starch and protein chemistry to the study of one of the most significant pathways of plant metabolism. The knowledge generated has potential application to the tailoring of starches for specific applications in the food and industrial sectors.