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Jun 2019

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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    

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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+), NADH (NADH), Stress (压力), Aluminum (), Arabidopsis (拟南芥), 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

  1. 96-well plate (Tarsons Product, India)

  2. 50 mL centrifuge tubes (Tarsons Product, India)

  3. 1.5/2 mL tubes (Tarsons Product, India)

  4. Root sample of Arabidopsis genotype Col-0

  5. Double distilled water

  6. Planton box (Tarsons Product, catalog number: 020080, size: 75 × 75 × 100mm)

  7. Sodium hypochlorite (NaOCl) (Himedia Laboratories, catalog number: PCT1311-5X50M)

  8. Calcium chloride (CaCl2) (Himedia Laboratories, catalog number: PCT0004-500G)

  9. Aluminum chloride (AlCl3) (Merck, catalog number: 8010810100)

  10. Nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, catalog number: NAD100-RO-1G)

  11. Nicotinamide adenine dinucleotide hydrogen (NADH) (Sigma-Aldrich, catalog number: 10107735001-500MG)

  12. Magnesium sulphate heptahydrate (MgSO4·7H2O) (Himedia Laboratories, catalog number: RM684-5KG)

  13. Manganese (II) Sulphate pentahydrate (MnSO4·5H2O) (FUJIFILM Wako Pure Chemical Corporation, catalog number:139-00825)

  14. Ferrous sulphate heptahydrate (FeSO4·7H2O) (Himedia Laboratories, catalog number: GRM3917-500G)

  15. Zinc sulphate hepta hydrate (ZnSO4·7H2O) (Himedia Laboratories, catalog number: PCT0118-1KG)

  16. Copper (II) sulphate pentahydrate (CuSO4·5H2O) (Himedia Laboratories, catalog number: RM630-500G)

  17. Potassium nitrate (KNO3) (Himedia Laboratories, catalog number: RM1401-500G)

  18. Boric acid (H3BO3) (Himedia Laboratories, catalog number: MB007-1KG)

  19. Sodium phosphate monobasic anhydrous (NaH2PO4) (Himedia Laboratories, catalog number: MB183-500G)

  20. Ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O) (Sigma-Aldrich, catalog number: 431346)

  21. Cobalt (II) chloride hexahydrate (CoCl2·6H2O) (Himedia Laboratories, catalog number: PCT0103-500G)

  22. EDTA, disodium salt hydrate (Na2EDTA) (Sigma-Aldrich, catalog number: E5134)

  23. Sodium nitrate (NaNO3) (Himedia Laboratories, catalog number: GRM1184-500G)

  24. Sodium phosphate monobasic dihydrate (NaH2PO4·2H2O) (Sigma-Aldrich, catalog number: 71505)

  25. Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O) (Sigma-Aldrich, catalog number: 71649)

  26. Calcium chloride dihydrate (CaCl2·2H2O) (Himedia Laboratories, catalog number: MB034-500G)

  27. Sodium hydroxide pellets (NaOH) (Himedia Laboratories, catalog number: MB095-500G)

  28. Hydrochloric acid (HCl) (Himedia Laboratories, catalog number: AS004-2.5L)

  29. Tris base (Sigma-Aldrich, catalog number: T1503)

  30. Bicine (Sigma-Aldrich, catalog number: B3876)

  31. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: 1.11714-1G)

  32. 1-Methoxy-5-methylphenazinium methyl sulfate (1-methoxy PMS) (Sigma-Aldrich, catalog number: M8640-100MG)

  33. Alcohol dehydrogenase (ADH), from Yeast (Sigma-Aldrich, catalog number: A7011)

  34. 0.2 M NaOH solution

  35. Modified MGRL solution (see Recipe 1)

  36. Bicine/NaOH buffer (see Recipe 2)

  37. 1 M Tris-HCl (see Recipe 3)

  38. 10 M Ethanol (see Recipe 4)

  39. 80 mM EDTA-2Na (see Recipe 5)

  40. ADH solution (see Recipe 6)

  41. Reaction Mixture (see Recipe 7)

  42. NAD standard (see Recipe 8)

  43. NADH standard (see Recipe 9)

Equipment

  1. Weighing balance (Sartorious, 0.1 mg–220 g)

  2. Pipettes/multi-channel pipette (Gilson, Pipettman, 2-2020-200 and 100–1000 µL)

  3. pH meter (pH Tutor, Eutech Instrument)

  4. Centrifuge (Eppendorf 5424 Microcentrifuge)

  5. Magnetic stirrer with hot plate (Tarsons Product, India)

  6. Micro pestle (Tarsons Product, India)

  7. Autoclave (Equitron, Equitron Medica Pvt. Ltd., India)

  8. Water bath (Equitron unstirred water bath, Equitron Medica Pvt. Ltd., India)

  9. pH test paper (Himedia Laboratories, India)

  10. Microtiter plate reader (SUNRISE microplate reader, TECAN)

  11. Nylon mesh (100 µM pore size)

  12. Fuji film plastic mounts, 35 mm (Fuji photo Co. Ltd. Japan)

Procedure

  1. 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).

  2. 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.


  3. 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.

  4. 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.

  5. 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

  1. MGRL solution

    Sr. No. Chemical constituents

    Solution

    Stock Conc.

    Solution

    Final 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  1. Apel, K. and Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55: 373-399.
  2. Awasthi, J. P., Saha, B., Panigrahi, J., Yanase, E., Koyama, H. and Panda, S. K. (2019). Redox balance, metabolic fingerprint and physiological characterization in contrasting North East Indian rice for Aluminum stress tolerance. Sci Rep 9(1): 8681.
  3. Dutilleul, C., Garmier, M., Noctor, G., Mathieu, C., Chetrit, P., Foyer, C. H. and de Paepe, R. (2003). Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation. Plant Cell 15(5): 1212-1226.
  4. Dutilleul, C., Lelarge, C., Prioul, J. L., De Paepe, R., Foyer, C. H. and Noctor, G. (2005). Mitochondria-driven changes in leaf NAD status exert a crucial influence on the control of nitrate assimilation and the integration of carbon and nitrogen metabolism. Plant Physiol 139(1): 64-78.
  5. Foyer, C. H. and Noctor, G. (2005). Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17(7): 1866-1875.
  6. Hampp, R., Goller, M. and Fullgraf, H. (1984). Determination of compartmented metabolite pools by a combination of rapid fractionation of oat mesophyll protoplasts and enzymic cycling. Plant Physiol 75(4): 1017-1021.
  7. Mittler, R., Vanderauwera, S., Gollery, M. and Van Breusegem, F. (2004). Reactive oxygen gene network of plants. Trends Plant Sci 9(10): 490-498.
  8. Moller, I. M. (2001). PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron Transport, NADPH Turnover, and Metabolism of Reactive Oxygen Species. Annu Rev Plant Physiol Plant Mol Biol 52: 561-591.
  9. Takita, E., Koyama, H. and Hara, T. (1999). Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.). Plant and Cell Physiology 40(5): 489-495.

简介

[摘要] 烟酰胺腺嘌呤二核苷酸 (NAD) 是在所有活细胞中发现的众多酶促反应的重要辅助因子。吡啶核苷酸(NAD +和 NADH)也是通过活性氧(ROS)发出信号的关键参与者,对植物中产生 ROS 和消耗 ROS 系统的调节至关重要。 NAD 含量是代谢整合、蛋白质去乙酰化和 DNA 修复的强大调节剂。 NAD 氧化和还原形式之间的平衡,即。 e .,NADH/NAD +比值,表示细胞的氧化还原状态,是反映细胞代谢健康的量度。在这里,我们提出了一种简单的方法来酶促估计 NAD +和 NADH 含量,使用醇脱氢酶 (ADH)、氧化还原酶和 MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5 -二苯基-2H-溴化四唑鎓)作为底物,1-甲氧基PMS(1-甲氧基-5-甲基吩嗪鎓甲基硫酸盐)作为电子载体。 MTT 被还原为紫色甲臜,然后被检测到。我们使用暴露于铝毒性和未经处理的对照条件下的拟南芥叶子样品。 NADH/NAD +连接新陈代谢的许多方面,并在植物发育过程和应激反应中发挥重要作用。因此,确定 NADH/NAD +在压力下的状态至关重要。


[背景] 烟酰胺腺嘌呤二核苷酸 (NAD) 是一种重要的辅酶,广泛存在于所有活细胞中。 NAD 的氧化和还原形式之间的平衡(NADH/NAD +比率)对细胞存活至关重要。该比率是指示细胞氧化还原状态的重要组成部分,对信号转导和表观遗传学等主要细胞过程很重要,并反映了代谢活动和细胞的健康状况。 NAD +负责代谢过程中分子之间的电子转移;因此,其水平对于维持正常的细胞呼吸功能至关重要。此外, NAD 在调节细胞氧化还原状态和控制信号传导和转录事件方面发挥作用(Awasthi等人,2019)。
细胞中NAD的消耗是细胞死亡的主要原因。量化吡啶核苷酸 NADH 和 NAD +的产生和消耗对于监测酶反应或筛选这些酶反应的调节剂或产物很重要。吡啶核苷酸参与其他防御和信号反应,例如一氧化氮的产生和反应性脂质衍生物的代谢。 NAD 状态可以改变光合作用和植物应激反应( Dutilleul 等。 , 2003), 表明 NAD 含量是代谢整合的强大调节剂 ( Dutilleul 等。 , 2005)。 NADH 和 NAD + 也是通过活性氧 (ROS) 发出信号的关键参与者(Moller,2001; Apel和 Hirt,2004; Mittler 等。 , 2004;门厅和诺克特,2005 年)。 NAD 消耗反应在应激条件下对于与 ROS 和其他氧化还原成分相互作用的信号传导很重要。这些核苷酸的氧化和还原速率的平衡是分解代谢和合成代谢过程继续进行的先决条件。因此, NADH/NAD + 比率是植物细胞代谢状态的代表,确定其在胁迫下的含量是了解胁迫反应机制的基础。

关键字:NAD+, NADH, 压力, 铝, 拟南芥, 氧化还原状态

材料和试剂
1. 96 孔板(印度Tarsons产品)
2. 50 mL 离心管(印度Tarsons产品)
3. 1.5/2 mL 管(印度Tarsons产品)
4. 拟南芥基因型 Col-0的根样本
5. 双蒸水
6. 普朗顿盒( Tarsons产品,目录号:020080,尺寸:75 × 75 × 100mm)
7. 次氯酸钠( NaOCl )( Himedia Laboratories,目录号:PCT1311-5X50M)
8. 氯化钙(CaCl 2 )( Himedia Laboratories,目录号:PCT0004-500G)
9. 氯化铝(AlCl 3 )(Merck,目录号:8010810100)
10. 烟酰胺腺嘌呤二核苷酸(NAD)(Sigma-Aldrich,目录号:NAD100-RO-1G)
11. 烟酰胺腺嘌呤二核苷酸氢(NADH)(Sigma-Aldrich,目录号:10107735001-500MG)
12. 七水硫酸镁(MgSO 4 · 7H 2 O)( Himedia Laboratories,目录号:RM684-5KG)
13. 五水合硫酸锰(II)(MnSO 4 · 5H 2 O)( FUJIFILM Wako Pure Chemical Corporation ,目录号:139-00825)
14. 硫酸亚铁(FeSO 4 · 7H 2 O)( Himedia Laboratories,目录号:GRM3917-500G)
15. 七水合硫酸锌(ZnSO 4 · 7H 2 O)( Himedia Laboratories,目录号:PCT0118-1KG)
16. 五水合硫酸铜(II)(CuSO 4 · 5H 2 O)( Himedia Laboratories,目录号:RM630-500G)
17. 硝酸钾(KNO 3 )( Himedia Laboratories,目录号:RM1401-500G)
18. 硼酸(H 3 BO 3 )( Himedia Laboratories,目录号:MB007-1KG)
19. 无水磷酸二氢钠(NaH 2 PO 4 )( Himedia Laboratories,目录号:MB183-500G)
20. 四水合钼酸铵((NH4) 6 Mo 7 O 24 · 4H 2 O)(Sigma-Aldrich,目录号:431346)
21. 六水合氯化钴(II)(CoCl 2 · 6H 2 O)( Himedia Laboratories,目录号:PCT0103-500G)
22. EDTA,二钠盐水合物(Na 2 EDTA)(Sigma-Aldrich ,目录号:E5134)
23. 硝酸钠(NaNO 3 )( Himedia Laboratories,目录号:GRM1184-500G)
24. 磷酸二氢钠二水合物(NaH 2 PO 4 · 2H 2 O)(Sigma-Aldrich,目录号:71505)
25. 磷酸氢二钠十二水合物(Na 2 HPO 4 · 12H 2 O)(Sigma-Aldrich,目录号:71649)
26. 氯化钙二水合物(CaCl 2 · 2H 2 O)( Himedia Laboratories,目录号:MB034-500G)
27. 氢氧化钠颗粒(NaOH)( Himedia Laboratories,目录号:MB095-500G)
28. 盐酸(HCl)( Himedia Laboratories,目录号:AS004-2.5L)
29. Tris碱基(Sigma-Aldrich,目录号:T1503)
30. Bicine(Sigma-Aldrich,目录号:B3876)
31. 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-溴化四唑(MTT)(Sigma-Aldrich,目录号:1.11714-1G)
32. 1-甲氧基-5-甲基吩嗪鎓甲基硫酸盐(1-甲氧基PMS)(Sigma-Aldrich,目录号:M8640-100MG)
33. 酒精脱氢酶(ADH),来自酵母(Sigma-Aldrich,目录号:A7011)
34. 0.2 M NaOH 溶液
35. 改进的 MGRL 解决方案(见配方 1)
36. Bicine/NaOH 缓冲液(见配方 2)
37. 1 M Tris-HCl(见配方 3)
38. 10 M 乙醇(见配方 4)
39. 80 mM EDTA-2Na(见配方 5)
40. ADH 溶液(见配方 6)
41. 反应混合物(见配方 7)
42. NAD 标准(见配方 8)
43. NADH 标准(见配方 9)


设备


1. 天平 ( Sartorious , 0.1 mg – 220 g)
2. 移液器/多道移液器(Gilson、 Piettman 、2-2020-200 和 100 – 1000 µL)
3. pH计(pH Tutor、 Eutech Instrument)
4. 离心机( Eppendorf 5424 微型离心机)
5. 带热板的磁力搅拌器(印度Tarsons产品)
6. 微型研杵(印度Tarsons产品)
7. 高压灭菌器( Equitron , Equitron Medica Pvt. Ltd.,印度)
8. 水浴( Equitron 非搅拌水浴, Equitron Medica Pvt. Ltd.,印度)
9. pH 试纸( Himedia Laboratories,印度)
10. 微量滴定板阅读器(SUNRISE 微孔板阅读器,TECAN)
11. 尼龙网(100 µM 孔径)
12. 富士胶片塑料卡口,35 毫米(日本富士照片有限公司)


程序


1. 在 1.5 mL 离心管中用 1% 次氯酸钠对可行的拟南芥种子进行表面消毒 3 分钟,并在蒸压蒸馏水中冲洗五次。在层流罩内执行所有程序以避免污染。将冲洗过的种子保持在 4°C,以便在黑暗条件下进行春化。 2 天后,将春化种子放在安装在 Fujifilm 塑料支架上的尼龙网上,让它们在无菌条件下漂浮在已填充改性 MGRL 溶液(配方 1)的Planton盒上,温度为 20 ± 2°C,用光周期为 14 h,光子通量密度为 220 μmol m -2 sec -1 (PAR)。 5天后,用一只手倒出改性的MGRL并用处理溶液(含有100 μM C aCl 2 ,pH 5.0的10 μM AlCl 3溶液)代替;更换溶液时,使用镊子将 Fujifilm 塑料支架与承载幼苗的网状物保持在一起。在处理开始后 6 和 12 小时采集样品(全植物组织)用于氧化还原池测定(图 1)。
2. 在 1.5 mL 离心管中用微杵在液氮中研磨样品(全植物组织,100 mg),然后用 1 mL 0.2 N HCl 提取。将匀浆在 16,000 × g和 4°C 下离心 10 分钟;制作多个等分的上清液(每个 0.2 mL)用于重复。


 
图1。 在 MGRL 水培溶液上无菌条件下生长的拟南芥植物。


3. 对于 NAD+ 测定,将 0.2 mL 提取物在沸水 (98–100°C) 中孵育 1 分钟,然后快速冷却并加入 20 µL 0.2 M NaH 2 PO 4 (pH 5.6) 中和,然后加入逐步添加 0.2 M NaOH 等分试样。每次添加后涡旋样品并用 pH 试纸检查 pH。最终的 pH 值应介于 5 和 6 之间,这需要大约 0.16 mL 的0.2 M NaOH。
4. 要测量 NADH,请提取 叶样品与 NAD +相同,但使用 0.2 M NaOH 作为萃取介质,并用 0.2 N HCl 中和加热的上清液等分试样,使所有样品的最终 pH 值为7 – 8。这需要大约 0.14 mL 的 0.2 N HCl。每次添加后涡旋样品,并用 pH 试纸验证 pH。
5. 制备酶反应混合物如下:
a. 在单独的试管中加入 MTT 和 1-甲氧基 PMS 并溶解在水中(在室温下制备这些溶液)(参见配方 7)。
b. 在 50 mL 离心管中加入 2 mL 的 1 M Bicine/NaOH 缓冲液、0.4 mL 的 1 M tris、1 mL 的 80 mM EDTA 和 1 mL 的 10 M 乙醇(参见配方 7)。
c. 将溶解的 MTT 和 1-甲氧基 PMS 添加到 50 mL 离心管中,将最终体积调整至 20 mL,并在 25°C 的水浴中孵育直至进一步使用(参见配方 7)。该溶液将作为反应混合物。
d. 准备 ADH 溶液并将其放在冰上(参见配方 6)。 
e. 将 40 μL每个标准样品(NAD +参见配方 8 ,NADH 参见配方 9)、植物样品(NAD +来自步骤 3和 NADH 来自步骤 4)和空白样品(40 μL水)加入 96 孔盘子。
f. 将 ADH (4 µL) 添加到反应混合物 (156 µL) 中并轻轻混合。
g. 在 96 孔板的每个样品孔中加入 160 μL 的酶反应混合物,并立即使用微量滴定板读取器测量吸光度。
h. 将测量吸光度的参数设置为:测量滤光片,570 nm;和动力学,以 1 分钟间隔进行 10 次测量,每次读数前摇动 5 秒。
i. 绘制 NAD +和 NADH 的标准图,并进一步评估植物样本内容(图 2)。


 
图 2。 NAD +的标准曲线 (A)和 NADH (B) 。在 570 nm 处测量吸光度。 


数据分析


所有分析和绘图均使用 Microsoft Office Excel 2016 电子表格完成。每个实验重复三次,提供的数据是平均值±标准误差 (SE)。使用单因素方差分析测试显着性。进行Duncan的多范围检验 (DMRT) 以在一组实验之间进行比较(图 3)。


 
图 3。 拟南芥 WT (Col-0) 根样品中 NAD +和 NADH含量及其比例的示例。
+和 NADH 及其比率进行绝对定量。值是三个独立实验的平均值 ± SE (n = 3)。根据 Duncan的多重范围检验,相同字母表示的平均值在 P < 0.05 时差异不显着。


食谱


1. MGRL解决方案
先生号 化学成分 解决方案
库存浓度 解决方案
最终浓度 制备 1 L 溶液所需的体积,pH 5.8
1 MgSO 4 · 7H 2 O 0.15 M 0.03 毫米 200 µL
2 Mn SO 4 · 5H 2 O 1.03 毫米 0.206 µM 200 µL
3 FeSO 4 · 7H 2 O 0.86 毫米 0.172 µM 200 µL
4 ZnSO 4 · 7H 2 O 0.1 毫米 0.02 µM 200 µL
5 CuSO 4 · 5H 2 O 0.1 毫米 0.02 µM 200 µL
6 硝酸钾3 0.3M 0.06 毫米 200 µL
7 H 3硼3 3.0毫米 0.6 µM 200 µL
8 (NH4)6Mo 7 O 24 · 4H 2 O 2.4微米 0.48纳米 200 µL
9 CoCl 2 · 6H 2 O 13微米 2.6纳米 200 µL
10 Na 2 EDTA 6.7 毫米 1.34 微米 200 µL
11 NaNO 3 0.4M 80微米 200 µL
12 Na-PO 4 (pH 5.8)
NaH 2 PO 4 · 2H 2 O
Na 2 HPO 4 · 12H 2 O
0.175 M
0.175 M
0.035 毫米
0.035 毫米 200 µL
13 CaCl 2 · 2H 2 O 1M 200微米 200 µL
根据样本量和植物种类准备足量的营养液;将 pH 值调至 5.8。


2. 1 M Bicine/NaOH (pH 8.0) 缓冲液
a. 将 16.317 g Bicine (MW = 163.17 g/mol]) 溶解在 75 mL 蒸馏水中
b. 使用 10 N NaOH 调节至 pH 8.0
c. 2 O填充至 100 mL 的最终体积
d. 过滤灭菌(推荐)或高压灭菌
e. 储存于 4 °C


3. 1 M Tris-HCl
a. 将 12.1 g Tris Base (TRIZMA) 溶解在 70 mL 蒸馏水中,加入浓 HCl 至 pH 8.0
b. 用蒸馏水填充至体积 1 L
c. 在室温下储存。


4. 10 M 乙醇
制备该溶液时,取 58.4 mL 无水乙醇,用蒸馏水补足 100 mL。


5. 80 毫米 EDTA-2Na
a. 将 29.77 g Na 2 EDTA 溶解在 80 mL 蒸馏水中并用 NaOH 将 pH 值调节至 8.0
b. 用蒸馏水将体积调整至 100 mL,在磁力搅拌器上剧烈搅拌,并在 4 °C下储存以延长储存时间。
c. 通过添加 NaOH 将溶液的 pH 值调节至 8.0,以完全溶解 Na 2 EDTA。


6. ADH 解决方案
将 8 mg ADH 添加到 1.5 mL 管中,并溶解在 1 mL 的 bicine/NaOH 中。溶解后,立即置于冰上备用。


7. 反应混合物制备
化学成分 全部的 20 毫升 最终浓度
MTT 毫克 3.48(溶于 6 mL 水中) 0.42 毫米
1-甲氧基经前综合症 毫克 3.72(溶于 6 mL 水中) 0.55 毫米
1 M Bicine/NaOH 毫升 2 0.1M
1 M Tris 毫升 0.4 20毫米
80 毫米 EDTA-2Na 毫升 1 4毫米
10 M 乙醇 毫升 1 0.5M
H 2 O ( MilliQ ) 毫升 3.6


8. NAD标准:NAD +标准
标准曲线( pmol /mL) 空白的 50 100 150 200 250 300 350 400
1 µM NAD + (µL) 0 5 10 15 20 25 30 35 40
H 2 O ( MilliQ ) (µL) 100 95 90 85 80 75 70 65 60
从每个浓度中取 40 μL 的样品。


9. NADH标准:NADH 标准
标准曲线( pmol /mL) 空白的 10 20 40 60 80 100 120 140
100 nM NADH (µL) 0 10 20 40 60 80 100 12(1 µM 库存) 14
H 2 O ( MilliQ ) (µL) 100 90 80 60 40 20 0 88 86
从每个浓度中取 40 µL 样品。


致谢


该协议改编自Hampp 等。 (1984)和泷田 等。 (1999)。


利益争夺 


作者声明没有利益冲突或竞争利益。


参考


1. Apel , K. 和 Hirt, H. (2004)。活性氧:新陈代谢、氧化应激和信号转导。 Annu Rev 植物生物学55:373-399。
2. Awasthi, JP, Saha , B., Panigrahi , J., Yanase, E., Koyama, H. 和 Panda, SK (2019)。对比东北印度水稻对铝胁迫的耐受性的氧化还原平衡、代谢指纹和生理特征。 科学代表9(1):8681。
3. Dutilleul , C., Garmier , M., Noctor , G., Mathieu, C., Chetrit, P., Foyer, CH 和 de Paepe , R. (2003)。叶线粒体调节全细胞氧化还原稳态,设置抗氧化能力,并通过改变信号和昼夜调节来确定抗逆性。 植物细胞15(5):1212-1226。
4. Dutilleul , C., Lelarge , C., Prioul , JL, De Paepe , R., Foyer, CH 和Noctor , G. (2005)。线粒体驱动的叶片 NAD 状态变化对硝酸盐同化的控制和碳和氮代谢的整合产生了至关重要的影响。 植物生理学139(1):64-78。
5. 门厅,CH 和Noctor ,G.(2005 年)。氧化还原稳态和抗氧化信号:压力感知和生理反应之间的代谢界面。 植物细胞17(7):1866-1875。
6. Hampp , R.、 Goller , M. 和Fullgraf , H. (1984)。通过燕麦叶肉原生质体的快速分馏和酶循环相结合来确定分隔的代谢物池。 植物生理学 75(4):1017-1021。
7. Mittler , R., Vanderauwera , S., Gollery , M. 和 Van Breusegem , F. (2004)。植物的活性氧基因网络。 趋势植物科学9(10):490-498。
8. 穆勒,IM (2001)。植物线粒体和氧化应激:电子传递、NADPH 周转和活性氧物质的代谢。 Annu Rev Plant Physiol Plant Mol Biol 52:561-591。
9. Takita , E.、Koyama, H. 和 Hara, T. (1999)。利用胡萝卜 ( Daucus carota L.)细胞中的有机酸代谢。 植物和细胞生理学40(5):489-495。




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引用: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.
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