Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra), Mutiloako etorbidea z/g, 31192 Mutiloa, Nafarroa, Spain.
Plant Cell Physiol. 2012 Feb;53(2):433-44. doi: 10.1093/pcp/pcr193. Epub 2011 Dec 30.
ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme comprising two small and two large subunits that catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism assumes that post-translational redox modification of AGP in response to light is a major determinant of fine regulation of transitory starch accumulation. According to this view, under oxidizing conditions occurring during the night the two AGP small subunits (APS1) are covalently linked via an intermolecular disulfide bridge that inactivates the protein, whereas under reducing conditions occurring during the day NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this work we have analyzed changes in the redox status of APS1 during dark-light transition in leaves of plants cultured under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli redox-insensitive AGP (GlgC) in the chloroplast. We also characterized aps1 plants expressing a redox-insensitive, mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when plants were cultured under photo-oxidative conditions. We also found that starch accumulation rates during the light in leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore, the pattern of starch accumulation during illumination in leaves of APS1mut-expressing aps1 mutants was similar to that of APS1-expressing aps1 mutants at any light intensity. The overall data demonstrate that post-translational redox modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.
ADP-葡萄糖焦磷酸化酶（AGP）是一种异四聚体酶，由两个小亚基和两个大亚基组成，催化 ADP-葡萄糖与淀粉生物合成的连接。目前关于叶片淀粉代谢的模式假设是，AGP 的翻译后氧化还原修饰对光的响应是暂时淀粉积累精细调节的主要决定因素。根据这一观点，在夜间发生的氧化条件下，两个 AGP 小亚基（APS1）通过分子间二硫键共价连接，使蛋白质失活，而在白天发生的还原条件下，NADP-硫氧还蛋白还原酶 C（NTRC）依赖的 APS1 还原单体化激活酶。在这项工作中，我们分析了在不同光照强度下培养的植物叶片中，暗-光转换过程中 APS1 的氧化还原状态变化。此外，我们还对 ntrc 突变体和在叶绿体中表达大肠杆菌氧化还原不敏感 AGP（GlgC）的 aps1 突变体的淀粉含量进行了时间进程分析。我们还表征了在叶绿体中表达氧化还原不敏感、突变 APS1（APS1mut）形式的 aps1 植物，其中涉及形成分子间二硫键的高度保守 Cys81 残基被丝氨酸取代。我们发现，只有在植物在光氧化条件下培养时，才会发生非常温和、依赖 NTRC 的 APS1 单体化过程。我们还发现，在 ntrc 突变体和 GlgC 表达 aps1 突变体的叶片中，光照期间的淀粉积累率与野生型叶片相似。此外，在任何光照强度下，在表达 APS1mut 的 aps1 突变体的叶片中，淀粉积累的模式与表达 APS1 的 aps1 突变体相似。总的来说，数据表明，AGP 的翻译后氧化还原修饰对光的响应并不是拟南芥中暂时淀粉积累精细调节的主要决定因素。
