Post-translational regulation of starch synthesis in higher plants

该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。 功能性的变化来自于诸如直链淀粉与支链淀粉的比率、葡聚糖链长度、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聚蛋白复合物，由可逆的蛋白磷酸化催化。 最近，我们已经利用遗传学的方法来证明，淀粉合成酶IIa和淀粉分支酶IIb的突变有超出酶活性的损失的影响，修改的性质的异聚体蛋白质复合物的大小，结构和性质的淀粉颗粒的后果。 我们认为，蛋白质磷酸化是决定淀粉合成酶之间的物理关联的关键，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）确定相关蛋白质之间的相互作用域; B）鉴定磷酸化位点和体内条件，在所述磷酸化位点和条件下每种酶的磷酸化状态被修饰; c）鉴定负责淀粉合成酶的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结构域，并改变所得淀粉表型的结构。 这些研究将使我们能够开发一个模型的作用，翻译后修饰的调节淀粉合成和结构。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，遗传学以及淀粉和蛋白质化学结合在一起，以研究植物代谢的最重要途径之一。 所产生的知识有可能应用于定制淀粉在食品和工业部门的具体应用。

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.