Mechanisms of biosynthesis and transport of ADP-glucose in rice endosperm cells

植物在异养器官的淀粉体中积累储存淀粉，作为下一代的能量来源。我们利用储存淀粉，这是一种天然的和高度可获取的生物质，不仅作为热量来源，而且作为各种工业原料。因此，了解植物淀粉的生物合成是一个直接关系到能量保障和增加的关键问题。ADPglucose pyrophosphorylase （AGPase）是淀粉生物合成中的关键酶，可催化ADPglucose （ADPG）的合成。与其他植物不同，谷类植物在胚乳细胞的淀粉体和细胞质中有两种类型的agpase，因此在细胞质中合成ADPG并将其运输到淀粉体的特定系统。然而，该系统的生理意义仍有待确定。本研究的目的是为提高水稻产量构建新的分子基础，重点研究谷物特异性系统。从水稻种子cDNA混合物中分离到ADPG转运体cDNA。RT-PCR分析表明，mRNA主要在种子中积累。在大肠杆菌细胞中的表达表明，该膜蛋白需要其他宿主细胞或载体。利用水稻AGPases的6个亚基基因的器官特异性表达谱，估计了胞质型和可塑性型AGPases的亚基组成。从大肠杆菌中纯化了重组胞质酶和塑质酶，并对其动力学性质进行了研究。虽然通过碘染色预测胞浆型比胞浆型在体内活性更高，但动力学性质表明胞浆型比胞浆型活性更高。这表明水稻胚乳细胞中存在一种未知的胞质AGPase激活因子。

Plants accumulate storage starch in amyloplasts of heterotrophic organs as an energy source for the next generations. We have utilized the storage starch, which is a natural and highly accessible biomass, as not only calorie sources but also various industrial raw materials. Thus, to understand the plant starch biosynthesis is a critical issue that is directly linked to ensuring and increasing of energy. ADPglucose pyrophsophorylase (AGPase) is a key enzyme in the starch biosynthesis and catalyzes the synthesis of ADPglucose (ADPG). Unlike the other plants, cereal plants have two types of AGPases in amyloplasts and cytosol of the endosperm cells and therefore the specific system that synthesizes ADPG in cytosol and transports it to amyloplasts. The physiological significance of this system, however, remains to be defined. The aim in this study was to construct a novel molecular basis for the improvement of rice plant productivity, focusing on the cereal specific system.The cDNA for ADPG transporter was isolated from rice seed cDNA mixture. Analysis using RT-PCR showed that the mRNA predominantly accumulates in seeds. The expression system in Escherichia coli cells suggested that the other host cell or vector is required for this membrane protein.The subunit compositions for cytosolic and plastidic AGPases were estimated by the organ-specific gene expression profiles of six subunit genes for rice AGPases. The recombinant cytosolic and plastidic enzymes were purified from E. coli and the kinetic properties were investigated. Although, compared with the plastidic form, the cytosolic form was predicted to be more active in vivo by iodine staining, the kinetic properties showed that the plastidic form has a higher activity than the cytosolic form. This suggests that there is an unknown activating factor for the cytosolic AGPase in rice endosperm cells.