Enhanced turnover of transitory starch by expression of up-regulated ADP-glucose pyrophosphorylases in Arabidopsis thaliana
Enhanced turnover of transitory starch by expression of up-regulated ADP-glucose pyrophosphorylases in Arabidopsis thaliana
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DOI：
10.1016/j.plantsci.2005.07.019
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发表时间：
2006-01-01
期刊：
影响因子：
5.2
通讯作者：
Ito, H
中科院分区：
文献类型：
--
作者：
Obana, Y;Omoto, D;Ito, H
adp -葡萄糖焦磷酸化酶（AGPase）在高等植物中催化淀粉生物合成的第一步。先前的研究表明，马铃薯和拟南芥AGPases的两个大亚基突变导致酶的变抗特性上调，即对3-磷酸甘油酸（3-PGA）的敏感性升高和/或对无机磷酸盐（Pi）抑制的抗性升高。为了阐明这些突变组合对拟南芥变构和动力学特性的影响，我们将Ala33替换为Lys和Gly96替换为Asn，并将其植入拟南芥叶片中显性AGPase的大亚基ApL1中。当与野生型小亚基（wtApS1）在大肠杆菌中共表达时，得到的重组酶Up3表现出与含有单突变的Up1和Up2不同的变构调节特性。与野生型相比，Up3对3-PGA的亲和力增加了2.5倍，并且比其他上调酶对Pi的抗性更强。因此，Up3的变构调节特性是两个突变的非加性产物。为了研究ApL1上调对瞬时淀粉生物合成的影响，我们将野生型、Up1、Up2和Up3 Apt]基因导入到ApL1缺失的拟南芥TL46中。表达野生型Apt]基因的转基因植株将淀粉水平恢复到接近正常水平，表明引入的基因产物弥补了TL46植株的遗传缺陷。与野生型植物相比，表达上调Apt]基因的转基因植物表现出更高的AGPase活性和瞬时淀粉周转，后者导致碳和能量升高，以支持夜间异养生长。2005爱思唯尔爱尔兰有限公司版权所有。
ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch biosynthesis in higher plants. Previous studies have shown that two mutations in the large subunits of the potato and Arabidopsis AGPases resulted in the generation of enzymes with up-regulated allosteric properties, i.e. elevated sensitivity to 3-phosphoglycerate (3-PGA) and/or resistance towards inorganic phosphate (Pi) inhibition. To elucidate the effect of combining these mutations on the allosteric and kinetic properties, Ala33 to Lys and Gly96 to Asn substitutions were engineered into ApL1, the large subunit of the dominant Arabidopsis leaf AGPase expressed in leaves. When co-expressed with wild-type small subunit (wtApS1) in Escherichia coli, the resulting recombinant enzyme, designated Up3, showed allosteric regulatory properties distinct from Up1 and Up2 containing the single mutations. Up3 showed a 2.5-fold increase in 3-PGA affinity compared to wild-type and was more resistance to Pi than the other up-regulatory enzymes. Hence, the allosteric regulatory properties of Up3 are a non-additive product of the two mutations. To examine the effects of up-regulated ApL1 on transitory starch biosynthesis, the wild-type, Up1, Up2 and Up3 Apt] genes were introduced into Arabidopsis ApL1 deficient TL46. Transgenic plants expressing the wild-type Apt] gene restored starch levels to near normal levels, indicating that the introduced gene products complemented the genetic deficiency in TL46 plants. Transgenic plants expressing up-regulatory Apt] genes showed greater AGPase activity and turnover of transitory starch than wild-type plants, the latter resulting in elevated carbon and energy to support heterotrophic growth during the night. (c) 2005 Elsevier Ireland Ltd. All rights reserved.