Kruger Nicholas J, Masakapalli Shyam K, Ratcliffe R George
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
School of Basic Sciences, Indian Institute of Technology Mandi, Mandi, 175005, HP, India.
Methods Mol Biol. 2017;1670:1-16. doi: 10.1007/978-1-4939-7292-0_1.
Carbohydrates are the dominant respiratory substrate in many plant cells. However, the route of carbohydrate oxidation varies depending on the relative cellular demands for energy, reductant, and precursors for biosynthesis. During these processes individual substrate carbon atoms are differentially released as carbon dioxide by specific reactions in the network, and this can be measured by monitoring the release of CO from a range of positionally labeled forms of [C]glucose. Although the relative amounts of carbon dioxide produced from different carbon positions do not allow precise determination of fluxes, they are indicative of the route of carbohydrate utilization. Such information can be used to determine whether a comprehensive metabolic flux analysis is merited, and also to facilitate independent verification of flux maps generated by other techniques. This chapter describes an approach to determine and interpret the pattern of oxidation of carbohydrates by monitoring CO release during metabolism of exogenously supplied [1-C]-, [2-C]-, [3,4-C]-, and [6-C]glucose. The method is exemplified by studies on Arabidopsis cell suspension cultures, but the protocol can be easily adapted for the investigation of other plant materials.
碳水化合物是许多植物细胞中的主要呼吸底物。然而，碳水化合物的氧化途径会因细胞对能量、还原剂和生物合成前体的相对需求而有所不同。在这些过程中，单个底物碳原子通过网络中的特定反应以二氧化碳的形式差异释放，这可以通过监测一系列位置标记的[C]葡萄糖形式的CO释放来测量。尽管从不同碳位置产生的二氧化碳相对量无法精确确定通量，但它们可指示碳水化合物的利用途径。此类信息可用于确定是否值得进行全面的代谢通量分析，也有助于独立验证其他技术生成的通量图。本章描述了一种通过监测外源供应的[1-C]-、[2-C]-、[3,4-C]-和[6-C]葡萄糖代谢过程中的CO释放来确定和解释碳水化合物氧化模式的方法。该方法以拟南芥细胞悬浮培养物的研究为例，但该方案可轻松适用于其他植物材料的研究。
