Adenine Nucleotide and Nicotinamide Adenine Dinucleotide Measurements in Plants

Youjun Zhang (1, 2) , Ina Krahnert (1) , Antje Bolze (1) , Yves Gibon (3) , Alisdair Fernie (4, 2)

1 MPI-MP - Max-Planck-Institut für Molekulare Pflanzenphysiologie
2 CPSBB - Center of Plant Systems Biology and Biotechnology [Plovdiv]
3 BFP - Biologie du fruit et pathologie
4 MPI-MP - Max Planck Institute of Molecular Plant Physiology

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 re-dox 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 metabo-lites. © 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 Keywords: ADP r ATP r NAD + /NADP + r NADH/NADPH

Mots clés: ADP; ATP; NAD+/NADP+; NADH/NADPH

Mots clés: Métabolisme; Plante; Physiologie végétale

Domaines: Sciences du Vivant [q-bio]; Biologie végétale; Amélioration des plantes

Youjun Zhang, Ina Krahnert, Antje Bolze, Yves Gibon, Alisdair Fernie. Adenine Nucleotide and Nicotinamide Adenine Dinucleotide Measurements in Plants. Current Protocols in Plant Biology, 2020, 5 (3), pp.e20115. ⟨10.1002/cppb.20115⟩. ⟨hal-02926938⟩