Adenine Nucleotide and Nicotinamide Adenine Dinucleotide Measurements in Plants.

Q1 Agricultural and Biological Sciences Current protocols in plant biology Pub Date : 2020-09-01 DOI:10.1002/cppb.20115

Youjun Zhang, Ina Krahnert, Antje Bolze, Yves Gibon, Alisdair R Fernie

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引用次数: 11

Abstract

As the principal co-factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox-active substrate for multiple catabolic and anabolic reactions with the ratios of NAD⁺ /NADH and NADP⁺ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling-based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD⁺ /NADP⁺ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches.

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植物中腺嘌呤核苷酸和烟酰胺腺嘌呤二核苷酸的测定。

作为许多代谢途径的主要辅助因子，腺嘌呤核苷酸和烟酰胺腺嘌呤二核苷酸的测量提供了细胞能量代谢的重要信息。然而，由于它们的快速和可逆转化以及它们相对较低的浓度范围，很难测量这些化合物。在这里，我们描述了一种高灵敏度和选择性的离子配对高效液相色谱荧光检测方法来定量植物中的腺嘌呤核苷酸。此外，烟酰胺腺嘌呤二核苷酸是多种分解代谢和合成代谢反应中至关重要的氧化还原活性底物，NAD+ /NADH和NADP+ /NADPH的比值被认为是细胞内氧化还原电位和代谢状态的指标。在这里，我们描述了高度敏感的酶循环比色法(检测限在pmol范围内)，在涉及酸或碱提取的简单提取程序之后进行，以允许测量这些代谢物的细胞水平。©2020作者。基本方案1:准备用于测量的植物材料基本方案2:通过HPLC测量ATP, ADP和AMP基本方案3:NAD+ /NADP+测量基本方案4:NADH/NADPH测量基本方案5:数据分析和质量控制方法。

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Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.