Ma Fangfang, Jazmin Lara J, Young Jamey D, Allen Doug K
Donald Danforth Plant Science Center, St. Louis, MO, USA.
Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
Methods Mol Biol. 2017;1653:167-194. doi: 10.1007/978-1-4939-7225-8_12.
Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary C metabolic flux analysis (INST-MFA), which is based on transient labeling studies at metabolic steady state, offers a comprehensive platform to quantify plant central metabolism. In this chapter, we describe the application of INST-MFA to investigate metabolism in leaves. Leaves are an autotrophic tissue, assimilating CO over a diurnal period implying that the metabolic steady state is limited to less than 12 h and thus requiring an INST-MFA approach. This strategy results in a comprehensive unified description of photorespiration, Calvin cycle, sucrose and starch synthesis, tricarboxylic acid (TCA) cycle, and amino acid biosynthetic fluxes. We present protocols of the experimental aspects for labeling studies: transient CO labeling of leaf tissue, sample quenching and extraction, mass spectrometry (MS) analysis of isotopic labeling data, measurement of sucrose and amino acids in vascular exudates, and provide details on the computational flux estimation using INST-MFA.
光呼吸是光合作用的核心组成部分；然而，为了更好地理解其作用，应将其置于一个综合代谢网络的背景下看待，而不是一系列独立运行的单个反应。基于代谢稳态下的瞬态标记研究的非稳态碳代谢通量分析（INST-MFA），为量化植物中心代谢提供了一个全面的平台。在本章中，我们描述了INST-MFA在研究叶片代谢中的应用。叶片是一种自养组织，在一个昼夜周期内同化二氧化碳，这意味着代谢稳态仅限于不到12小时，因此需要采用INST-MFA方法。这种策略能够对光呼吸、卡尔文循环、蔗糖和淀粉合成、三羧酸（TCA）循环以及氨基酸生物合成通量进行全面统一的描述。我们介绍了标记研究实验方面的方案：叶片组织的瞬态CO标记、样品淬灭与提取、同位素标记数据的质谱（MS）分析、维管束渗出物中蔗糖和氨基酸的测定，并提供了使用INST-MFA进行计算通量估计的详细信息。
