铝胁迫下植物样品氧化还原池(NAD<sup>+</sup>， NADH含量)的定量分析

Jay Prakash Awasthi; Bedabrata Saha; Hiroyuki Koyama; Sanjib  Kumar Panda

doi:10.21769/BioProtoc.4444

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

铝胁迫下植物样品氧化还原池(NAD⁺， NADH含量)的定量分析

Jay Prakash Awasthi email

BS Bedabrata Saha

HK Hiroyuki Koyama

SP Sanjib Kumar Panda

发布: 2022年06月20日第12卷第12期 DOI: 10.21769/BioProtoc.4444 浏览次数: 2367

评审: Khyati Hitesh ShahAbhijit Arun DasputeTaraka Ramji Moturu

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Abstract

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.

Keywords: NAD⁺ (NAD⁺)

Redox status (氧化还原状态)

Background

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme ubiquitously found in all living cells. The balance between the oxidized and reduced forms of NAD (the NADH/NAD⁺ ratio) is crucial to cell survival. This ratio is an important component that indicates the redox state of a cell, important for major cellular processes like signal transduction and epigenetics, and reflects both the metabolic activities and the health of cells. NAD⁺ is responsible for the transfer of electrons between molecules during metabolic processes; therefore, its levels are essential for maintaining normal cellular respiratory function. Furthermore, NAD functions in modulating cellular redox status and controlling signaling and transcriptional events (Awasthi et al., 2019).

Depletion of NAD in cells is a major cause of cell death. Quantifying the generation and consumption of pyridine nucleotides, NADH and NAD⁺, is important to monitor enzymatic reactions or screen the modulator or product of these enzyme reactions. Pyridine nucleotides are involved in other defense and signaling reactions, such as nitric oxide production and metabolism of reactive lipid derivatives. NAD status can alter photosynthesis and plant stress responses (Dutilleul et al., 2003), suggesting that NAD content is a powerful modulator of metabolic integration (Dutilleul et al., 2005). NADH and NAD⁺ are also key players in signaling through reactive oxygen species (ROS) (Moller, 2001; Apel and Hirt, 2004; Mittler et al., 2004; Foyer and Noctor, 2005). NAD-consuming reactions are of importance in stress conditions for signaling in interactions with ROS and other redox components. A balance in the rates of oxidation and reduction of these nucleotides is a prerequisite for the continuation of both catabolic and anabolic processes. Therefore, the NADH/NAD⁺ ratio is a proxy for the metabolic state of plant cells, and determining its content under stress is fundamental for understanding stress response mechanisms.

Materials and Reagents

96-well plate (Tarsons Product, India)
50 mL centrifuge tubes (Tarsons Product, India)
1.5/2 mL tubes (Tarsons Product, India)
Root sample of Arabidopsis genotype Col-0
Double distilled water
Planton box (Tarsons Product, catalog number: 020080, size: 75 × 75 × 100mm)
Sodium hypochlorite (NaOCl) (Himedia Laboratories, catalog number: PCT1311-5X50M)
Calcium chloride (CaCl₂) (Himedia Laboratories, catalog number: PCT0004-500G)
Aluminum chloride (AlCl₃) (Merck, catalog number: 8010810100)
Nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, catalog number: NAD100-RO-1G)
Nicotinamide adenine dinucleotide hydrogen (NADH) (Sigma-Aldrich, catalog number: 10107735001-500MG)
Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (Himedia Laboratories, catalog number: RM684-5KG)
Manganese (II) Sulphate pentahydrate (MnSO₄·5H₂O) (FUJIFILM Wako Pure Chemical Corporation, catalog number:139-00825)
Ferrous sulphate heptahydrate (FeSO₄·7H₂O) (Himedia Laboratories, catalog number: GRM3917-500G)
Zinc sulphate hepta hydrate (ZnSO₄·7H₂O) (Himedia Laboratories, catalog number: PCT0118-1KG)
Copper (II) sulphate pentahydrate (CuSO₄·5H₂O) (Himedia Laboratories, catalog number: RM630-500G)
Potassium nitrate (KNO₃) (Himedia Laboratories, catalog number: RM1401-500G)
Boric acid (H₃BO₃) (Himedia Laboratories, catalog number: MB007-1KG)
Sodium phosphate monobasic anhydrous (NaH₂PO₄) (Himedia Laboratories, catalog number: MB183-500G)
Ammonium molybdate tetrahydrate ((NH4)₆Mo₇O₂₄·4H₂O) (Sigma-Aldrich, catalog number: 431346)
Cobalt (II) chloride hexahydrate (CoCl₂·6H₂O) (Himedia Laboratories, catalog number: PCT0103-500G)
EDTA, disodium salt hydrate (Na₂EDTA) (Sigma-Aldrich, catalog number: E5134)
Sodium nitrate (NaNO₃) (Himedia Laboratories, catalog number: GRM1184-500G)
Sodium phosphate monobasic dihydrate (NaH₂PO₄·2H₂O) (Sigma-Aldrich, catalog number: 71505)
Sodium phosphate dibasic dodecahydrate (Na₂HPO₄·12H₂O) (Sigma-Aldrich, catalog number: 71649)
Calcium chloride dihydrate (CaCl₂·2H₂O) (Himedia Laboratories, catalog number: MB034-500G)
Sodium hydroxide pellets (NaOH) (Himedia Laboratories, catalog number: MB095-500G)
Hydrochloric acid (HCl) (Himedia Laboratories, catalog number: AS004-2.5L)
Tris base (Sigma-Aldrich, catalog number: T1503)
Bicine (Sigma-Aldrich, catalog number: B3876)
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: 1.11714-1G)
1-Methoxy-5-methylphenazinium methyl sulfate (1-methoxy PMS) (Sigma-Aldrich, catalog number: M8640-100MG)
Alcohol dehydrogenase (ADH), from Yeast (Sigma-Aldrich, catalog number: A7011)
0.2 M NaOH solution
Modified MGRL solution (see Recipe 1)
Bicine/NaOH buffer (see Recipe 2)
1 M Tris-HCl (see Recipe 3)
10 M Ethanol (see Recipe 4)
80 mM EDTA-2Na (see Recipe 5)
ADH solution (see Recipe 6)
Reaction Mixture (see Recipe 7)
NAD standard (see Recipe 8)
NADH standard (see Recipe 9)

Equipment

Weighing balance (Sartorious, 0.1 mg–220 g)
Pipettes/multi-channel pipette (Gilson, Pipettman, 2-2020-200 and 100–1000 µL)
pH meter (pH Tutor, Eutech Instrument)
Centrifuge (Eppendorf 5424 Microcentrifuge)
Magnetic stirrer with hot plate (Tarsons Product, India)
Micro pestle (Tarsons Product, India)
Autoclave (Equitron, Equitron Medica Pvt. Ltd., India)
Water bath (Equitron unstirred water bath, Equitron Medica Pvt. Ltd., India)
pH test paper (Himedia Laboratories, India)
Microtiter plate reader (SUNRISE microplate reader, TECAN)
Nylon mesh (100 µM pore size)
Fuji film plastic mounts, 35 mm (Fuji photo Co. Ltd. Japan)

Procedure

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Awasthi, J. P., Saha, B., Koyama, H. and Panda, S. K. (2022). Quantitative Analysis of Redox Pool (NAD⁺, NADH Content) in Plant Samples Under Aluminum Stress. Bio-protocol 12(12): e4444. DOI: 10.21769/BioProtoc.4444.