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首页> 外文期刊>Journal of Experimental Botany >Metabolic turnover analysis by a combination of in vivo13C-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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Metabolic turnover analysis by a combination of in vivo13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves

机译：结合 13 CO 2 的体内 13 C标记和CE-MS / MS进行代谢谱分析，可进行代谢转换分析烟草叶片C 3 光合途径的限制性步骤

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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.
机译：了解植物中代谢途径的控制要求直接测量代谢周转率。糖的磷酸代谢，包括卡尔文循环，是C 3 光合作用的主要途径，其动态状态尚未在高等植物的叶片中进行定量评估。由于叶片中CO 2 的固定速率影响光合碳代谢的通量，因此开发了一种新型的 13 C标记系统，该系统采用 13 CO 2 用于动力学测定代谢转换，这是每种代谢物中 13 C标记比例的时间过程。该系统配有气体交换室，该室可实时监测CO 2 的固定率；冷冻钳可切除标记的叶片，并同时通过液氮淬灭代谢反应。光合作用室。通过用毛细管电泳串联质谱法检测质量同位素异构体丰度来进行动力学测量。由于植物细胞中某些糖磷酸酯的数量极少，因此优化了多反应监测方法以测定每种化合物以进行灵敏检测。我们的分析系统能够监测新鲜烟草（Nicotiana tabacum）叶片中糖磷酸酯的体内转化，这表明葡萄糖-1-磷酸酯（G1P）的转化率显着低于其他糖磷酸酯（包括葡萄糖）的转化率-6-磷酸（G6P）。 G1P的池大小是G6P的池大小的12倍。这些结果表明，G6P向G1P的转化是糖磷酸途径中的限速步骤之一。

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