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首页> 外文期刊>Journal of Experimental Botany >Metabolic turnover analysis by a combination of in vivo p#pdC-labelling from p#pdCO and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C photosynthetic pathway in Nicotiana tabacum leaves.
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Metabolic turnover analysis by a combination of in vivo p#pdC-labelling from p#pdCO and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C photosynthetic pathway in Nicotiana tabacum leaves.

机译：通过体内p＃pdCO的p＃pdC-标记和CE-MS / MS的代谢谱分析相结合的代谢转换分析揭示了烟草叶片中C光合途径的限速步骤。

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摘要

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 C 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 CO fixation rate in leaves, a novel in vivo p#pdC-labelling system was developed with p#pdCO for the kinetic determination of metabolic turnover that was the time-course of the p#pdC-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO 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。由于光合作用碳代谢的通量受叶片中CO固定率的影响，因此开发了一种新型的p＃pdCO体内p＃pdC标记系统，用于动态测定代谢周转率，这是p＃的过程。每种代谢物中的pdC标记比率。该系统配备了一个气体交换室，该气体交换室能够实时监测CO的固定率;一个冷冻钳可切除标记的叶片，同时在光合作用室内通过液氮淬灭代谢反应。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非常少，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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