Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants
Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants
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DOI：
10.1007/978-1-4939-7225-8_12
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发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Allen, Doug K.
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文献类型：
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作者：
Ma, Fangfang;Jazmin, Lara J.;Allen, Doug K.
光呼吸是光合作用的核心组成部分；然而，为了更好地理解它的作用，应该在一个综合的代谢网络的背景下来看待它，而不是一系列独立运行的单独反应。同位素非平稳C-13代谢通量分析(Inst-MFA)建立在代谢稳态的瞬时标记研究基础上，为定量研究植物的中枢代谢提供了一个全面的平台。在本章中，我们描述了Inst-MFA在研究叶片代谢方面的应用。叶片是一种自养组织，在一天的一段时间内吸收二氧化碳，这意味着代谢的稳定状态被限制在12小时以内，因此需要INST-MFA方法。这一策略导致了对光呼吸、卡尔文循环、蔗糖和淀粉合成、三羧酸(TCA)循环和氨基酸生物合成通量的全面统一描述。我们介绍了用于标记研究的实验方面的方案：叶组织的瞬时(CO2)-C-13标记、样品猝灭和提取、同位素标记数据的质谱分析、维管分泌物中蔗糖和氨基酸的测量，并提供了使用Inst-MFA计算通量的详细信息。
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-13 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 CO2 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 (CO2)-C-13 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.