Quantitative Analysis of Redox Pool (NAD+, NADH Content) in Plant Samples Under Aluminum Stress

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor of numerous enzymatic reactions found in all living cells. Pyridine nucleotides (NAD+ and NADH) are also key players in signaling through reactive oxygen species (ROS), being crucial in the regulation of both ROS-producing and ROS-consuming systems in plants. NAD content is a powerful modulator of metabolic integration, protein de-acetylation, and DNA repair. The balance between NAD oxidized and reduced forms, i.e., the NADH/NAD+ ratio, indicates the redox state of a cell, and it is a measurement that reflects the metabolic health of cells. Here we present an easy method to estimate the NAD+ and NADH content enzymatically, using alcohol dehydrogenase (ADH), an oxido-reductase enzyme, and with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as the substrate and 1-methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate) as the electron carrier. MTT is reduced to a purple formazan, which is then detected. We used Arabidopsis leaf samples exposed to aluminum toxicity and under untreated control conditions. NADH/NAD+ connects many aspects of metabolism and plays vital roles in plant developmental processes and stress responses. Therefore, it is fundamental to determine the status of NADH/NAD+ under stress.