Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.
Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves.
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DOI：
10.1093/jxb/erp374
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发表时间：
2010-02
影响因子：
6.9
通讯作者：
Miyake C
中科院分区：
文献类型：
--
作者：
Hasunuma T;Harada K;Miyazawa S;Kondo A;Fukusaki E;Miyake C
理解植物中代谢途径的控制需要直接测量代谢周转率。糖磷代谢，包括卡尔文循环，是C3光合作用的主要途径，在高等植物叶片中，其动态状态尚未被定量评估。由于光合碳代谢的通量受叶片中CO2固定率的影响，因此开发了一种新的体内13 C标记系统，用于13 CO2代谢周转率的动力学测定，即每个代谢物中13 C标记率的时间过程。该系统配备了一个气体交换室，可以实时监测CO2固定率和一个冷冻夹，该冷冻夹可以切除标记的叶子，同时通过光合作用室内的液氮淬灭代谢反应。动力学测量进行检测质量同位素丰度与毛细管电泳-串联质谱。由于植物细胞中某些糖磷酸盐的量非常少，因此优化了多反应监测方法以测定每种化合物，从而进行灵敏检测。我们的分析系统使糖磷酸盐的体内营业额进行监测新鲜烟草（烟草）叶片，这表明，葡萄糖-1-磷酸（G1 P）的营业率显着低于其他糖磷酸盐，包括葡萄糖-6-磷酸（G6 P）。G1 P的池大小比G6 P小12倍。这些结果表明，G6 P转化为G1 P是糖磷酸途径中的限速步骤之一。
Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C3 photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO2 fixation rate in leaves, a novel in vivo 13C-labelling system was developed with 13CO2 for the kinetic determination of metabolic turnover that was the time-course of the 13C-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO2 fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.
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影响因子：
4.3
作者：
Lytovchenko, A;Sweetlove, L;Fernie, AR
通讯作者：
Fernie, AR
影响因子：
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KOSSMANN, J;SONNEWALD, U;WILLMITZER, L
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