Functional analysis of the starch related glucose 1-phosphate metabolism in higher plants

在过去的几年里，淀粉代谢的生物化学得到了极大的提高。这种复杂性与30多种进化保守的酶以及与淀粉合成和降解有关的各种碳通量的协同作用有关。最近，通过原位实验发现，葡萄糖1-磷酸直接转运到拟南芥叶肉细胞和马铃薯块茎细胞，进而转运到叶绿体和淀粉体中。此外，还证明了葡萄糖1-磷酸的摄取与淀粉翻转之间的直接联系。到目前为止，所涉及的转运蛋白的分子性质以及对淀粉代谢的具体重要性尚不清楚。这项建议使用了各种实验方法，并集中在以下几个方面：a)鉴定拟南芥和马铃薯中涉及的葡萄糖1-磷酸转运蛋白，包括对潜在转运蛋白的特异性进行详细分析。因此，将分析在酵母和/或非洲爪哇卵母细胞中表达的转运蛋白。b)将通过产生除潜在的葡萄糖1-磷酸转运蛋白之外缺乏淀粉相关酶的各种拟南芥突变体来分析葡萄糖1-磷酸转运蛋白对淀粉代谢的重要性。将分析不同突变体的碳通量和淀粉代谢，包括淀粉颗粒形态、淀粉内部结构和淀粉相关酶。c)在不同突变体中，将使用高灵敏度的葡萄糖1-磷酸和非水分离分析葡萄糖1-磷酸在不同器官、组织和隔室中的分布。

Over the last years the biochemistry of starch metabolism is massively increased. The complexity is related to a concerted action of more than 30 evolutionary conserved enzymes and of various carbon fluxes related to starch syntheses as well as degradation. Recently, a direct transport of glucose 1-phosphate into Arabidopsis mesophyll cells and potato tuber cells and further into plastids (chloroplasts and amyloplast) was identified using in situ experiments. Moreover, a direct link of the up taken glucose 1-phosphate and starch turn-over was demonstrated. The molecular nature of the involved transporters as well as the detailed importance for starch metabolism is so far unclear. This proposal uses various experimental approaches and focuses on the following aspects:a) The identification of the involved glucose 1-phosphate transporters in Arabidopsis as well as in potato, including a detailed analysis of the specificity of the potentially transporters. Therefore, the transporters will be analysed expressed in yeast and/or Xenopus oocytes.b) The importance of the glucose 1-phosphate transport for starch metabolism will be analyzed by generating various Arabidopsis mutants lacking starch related enzymes in addition to the potentially glucose 1-phosphate transporters. The various mutants will be analyzed in respect to carbon fluxes and starch metabolism, including starch granule morphology, starch internal structure and starch related enzymes.c) In the various mutants the distribution of glucose 1-phosphate in different organs, tissues, and compartments will be analyzed using a highly sensitive assay for glucose 1-phosphate and non-aqueous fractionation.