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

Surface sterilize viable Arabidopsis seeds in a 1.5 mL centrifuge tube with 1% Sodium hypochlorite for 3 min and rinse five times in autoclaved distilled water. Carry out all procedures inside a laminar flow hood to avoid contamination. Keep the rinsed seeds at 4°C for vernalization in dark conditions. After 2 days, place the vernalized seeds on the nylon mesh mounted on Fujifilm plastic mounts, and allow them to float on a Planton box already filled with modified MGRL solution (Recipe 1) in aseptic conditions, at 20 ± 2°C, with a photoperiod of 14 h, and with a photon flux density of 220 μmol m^-2 sec^-1 (PAR). After 5 days, with one hand decant the modified MGRL and replace with the treatment solution (10 μM AlCl₃ solution containing 100 μM CaCl₂, pH 5.0); while replacing the solution, hold the Fujifilm plastic mounts with the mesh bearing the seedlings on the other hand, using forceps. Harvest samples (whole plant tissue) for the redox pool assay at 6 and 12 h after the beginning of the treatment (Figure 1).
Grind samples (whole plant tissue, 100 mg) in liquid nitrogen with a micro pestle in a 1.5 mL centrifuge tube, and then extract with 1 mL of 0.2 N HCl. Centrifuged the homogenate at 16,000 × g and 4°C for 10 min; make multiple aliquots of the supernatant (0.2 mL each) for replicates.

Figure 1. Arabidopsis plant grown in aseptic condition on MGRL hydroponic solution.
For the NAD⁺ assay, incubate 0.2 mL of extract in boiling water (98–100°C) for 1 min, and then cool it rapidly and neutralize it by adding 20 µL of 0.2 M NaH₂PO₄ (pH 5.6), followed by the stepwise addition of 0.2 M NaOH aliquots. Vortex the sample after each addition and check pH with pH indicator paper. The final pH should be between 5 and 6, which requires approximately 0.16 mL of 0.2 M NaOH.
To measure NADH, extract leaf samples as for NAD⁺ but with 0.2 M NaOH as the extraction medium, and neutralize the heated supernatant aliquot with 0.2 N HCl to a final pH of 7–8 for all samples. This requires approximately 0.14 mL of 0.2 N HCl. Vortex the sample after each addition and verify pH with pH indicator paper.
Prepare the enzymatic reaction mixture as follows:
1. Add MTT and 1-Methoxy PMS in separate tubes and dissolve in water (prepare these solutions at room temperature) (see Recipe 7).
2. Add 2 mL of 1 M Bicine/NaOH Buffer, 0.4 mL of 1 M tris, 1 mL of 80 mM EDTA, and 1 mL of 10 M ethanol in a 50 mL centrifuge tube (see Recipe 7).
3. Add the dissolved MTT and 1-Methoxy PMS to the 50 mL centrifuge tube, adjust the final volume to 20 mL, and incubate in a water bath at 25°C until further use (see Recipe 7). This solution will act as the reaction mixture.
4. Prepare the ADH solution and keep it on ice (see Recipe 6).
5. Add 40 μL of each standard sample (see Recipe 8 for NAD⁺, 9 for NADH), plant sample (from step 3 for NAD⁺ and from step 4 for NADH), and blank sample (40 μL water) to a 96-well plate.
6. Add ADH (4 µL) to the reaction mixture (156 µL) and gently mix.
7. Add 160 µL of enzymatic reaction mixture into each sample well of the 96-well plate and immediately measure the absorbance using a microtiter plate reader.
8. Set the parameter for measurement of absorbance as: measurement filter, 570 nm; and kinetics, 10 measurements at 1 min intervals, shaking for 5 s before every reading.
9. Plot the standard graphs of NAD⁺ and NADH in a Microsoft Excel spreadsheet and further evaluate the plant sample contents (Figure 2).
Figure 2. Standard curve for NAD⁺ (A) and NADH (B). Absorbance measured at 570 nm.

Data analysis

All analysis and graph plotting was done using Microsoft Office Excel 2016 spreadsheets. Each experiment was repeated thrice and the data presented are mean ± standard error (SE). Significance was tested with one-way ANOVAs. Duncan’s multiple range test (DMRT) was performed for comparison among the set of experiments (Figure 3).

Figure 3. Example of NAD⁺ and NADH content and their ratio in Arabidopsis WT (Col-0) root samples.

Absolute quantification of NAD⁺ and NADH and their ratio using a microtiter plate reader coupled enzyme assay in different replicates (a, b, and c). Values are means ± SE (n = 3) of three separate experiments. Means denoted by the same letter were not significantly different at P < 0.05 according to Duncan’s multiple range test.

Recipes

MGRL solution

Sr. No.	Chemical constituents	SolutionStock Conc.	SolutionFinal conc.	Required volume for the preparation of 1 L solution, pH 5.8
1	MgSO₄·7H₂O	0.15 M	0.03 mM	200 µL
2	Mn SO₄·5H₂O	1.03 mM	0.206 µM	200 µL
3	FeSO₄·7H₂O	0.86 mM	0.172 µM	200 µL
4	ZnSO₄·7H₂O	0.1 mM	0.02 µM	200 µL
5	CuSO₄·5H₂O	0.1 mM	0.02 µM	200 µL
6	KNO₃	0.3 M	0.06 mM	200 µL
7	H₃BO₃	3.0 mM	0.6 µM	200 µL
8	(NH4)6Mo₇O₂₄·4H₂O	2.4 µM	0.48 nM	200 µL
9	CoCl₂·6H₂O	13 µM	2.6 nM	200 µL
10	Na₂EDTA	6.7 mM	1.34 µM	200 µL
11	NaNO₃	0.4 M	80 µM	200 µL
12	Na-PO₄ (pH 5.8) NaH₂PO₄·2H₂O Na₂HPO₄·12H₂O	0.175 M 0.175 M	0.035 mM 0.035 mM	200 µL
13	CaCl₂·2H₂O	1 M	200 µM	200 µL

Prepare adequate amounts of nutrient solution according to sample size and plant species; adjust pH to 5.8.

1 M Bicine/NaOH (pH 8.0) Buffer
1. Dissolve 16.317 g of Bicine (MW = 163.17 g/mol]) in 75 mL of distilled water
2. Adjust to pH 8.0 using 10 N NaOH
3. Fill to final volume of 100 mL with dH₂O
4. Filter sterilize (recommended) or autoclave
5. Store at 4°C
1 M Tris-HCl
1. Dissolve 12.1 g Tris Base (TRIZMA) in 70 mL of distilled water and add concentrated HCl to pH 8.0
2. Fill up to volume 1 L with distilled water
3. Store at room temperature.
10 M Ethanol
For the preparation of this solution, take 58.4 mL of absolute Ethanol and make up to 100 mL with distilled water.
80 mM EDTA-2Na
1. The dissolve 29.77 g of Na₂EDTA in 80 mL of distilled water and adjust the pH to 8.0 with NaOH
2. Adjust volume to 100 mL with distilled water, stir vigorously on a magnetic stirrer, and store at 4°C for longer storage.
3. Adjust the pH of the solution to 8.0 by the addition of NaOH to completely dissolve the Na₂EDTA.
ADH solution
Add 8 mg of ADH to a 1.5 mL tube and dissolve in 1 mL of bicine/NaOH. After dissolving, keep on ice for immediate use.

Reaction Mixture preparation

Chemicals constituents	Total	20 mL	Final concentration
MTT	mg	3.48 (dissolve in 6 mL of water)	0.42 mM
1-Methoxy PMS	mg	3.72 (dissolve in 6 mL of water)	0.55 mM
1 M Bicine/NaOH	mL	2	0.1 M
1 M Tris	mL	0.4	20 mM
80 mM EDTA-2Na	mL	1	4 mM
10 M EtOH	mL	1	0.5 M
H₂O (MilliQ)	mL	3.6

NAD standard: NAD⁺standard
Standard curve (pmol/mL) blank 50 100 150 200 250 300 350 400
1 µM NAD⁺(µL) 0 5 10 15 20 25 30 35 40
H₂O (MilliQ) (µL) 100 95 90 85 80 75 70 65 60
Take 40 µL of sample from each concentration.
NADH standard: NADH standard
Standard curve (pmol/mL) blank 10 20 40 60 80 100 120 140
100 nM NADH (µL) 0 10 20 40 60 80 100 12 (1 µM stock) 14
H₂O (MilliQ) (µL) 100 90 80 60 40 20 0 88 86
Take 40 µL of sample from each concentration.

Acknowledgments

This protocol was adapted from Hampp et al. (1984) and Takita et al. (1999).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

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