Abstract
Nitric oxide (NO), is a redox-active, endogenous signalling molecule involved in the regulation of numerous processes. It plays a crucial role in adaptation and tolerance to various abiotic and biotic stresses. In higher plants, NO is produced either by enzymatic or non-enzymatic reduction of nitrite and an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. There are several methods to measure NO production: mass spectrometry, tissue localization by DAF-FM dye. Electron paramagnetic resonance (EPR) also known as electron spin resonance (ESR) and spectrophotometric assays. The activity of NOS can be measured by L-citrulline based assay and spectroscopic method (NADPH utilization method). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). This experimental method describes visualization of NO using DAF-FM dye by fluorescence microscopy (Zeiss AXIOSKOP 2). The whole procedure is simplified, so it is easy to perform but has a high sensitivity for NO detection. In addition, spectrophotometry based protocols for assay of NOS, Nitrate Reductase (NR) and the content of S-nitrosothiols are also described. These spectrophotometric protocols are easy to perform, less expensive and sufficiently sensitive assays which provide adequate information on NO based regulation of physiological processes depending on the treatments of interest.
Keywords: Nitric oxide (NO) (一氧化氮), Nitric oxide synthase (NOS)-like enzyme (一氧化氮合酶（NOS）样蛋白), Nitrate Reductase (NR) (硝酸还原酶（NR）), Diaminofluorescein-FM (DAF-FM) (二氨基荧光素-FM（DAF-FM）), S-nitrosothiol (S-亚硝基硫醇)
Background
Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes like regulating synthesis of the cell wall (Correa-Aragunde et al., 2008; Xiong et al., 2009; Ye et al., 2015), ROS metabolism in plants (Delledonne et al., 2001), gene expression and regulation (Bogdan et al., 2000), programmed cell death (de Pinto et al., 2002), maturation and senescence (Yaacov et al., 1998). NO exerts a crucial role in protecting plants against various abiotic stresses (Hung et al., 2002). NO could significantly enhance antioxidative capacity by increasing the activities of catalase (CAT), ascorbate peroxidase (APX) and accumulating proline during wheat seed germination under osmotic stress (Zhang et al., 2003). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation (Yun et al., 2016)
Nitric oxide (NO) production in land plants classically involves two main routes: first, a reductive pathway involving both enzymatic and non-enzymatic reduction of nitrite into NO (Gupta et al., 2011); second, an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. Role of NR in NO production was suspected by low or no NR activity mutants which show no measurable NO. Later nia1/nia2 double mutants of Arabidopsis confirmed the role of NR in reduction of NO2- to NO in NADH dependent manner under both in vitro (Yamasaki et al., 1999) and in vivo (Vanin et al., 2004) condition. The possibility that NOS could catalyze NO synthesis in plants has also been a main controversial issue. Experimental evidence further increased suspicion about the existence of a plant NOS-like enzyme. It was reported that the L-citrulline based assay commonly used to measure a NOS activity in plant extracts is prone to artefacts (Tischner et al., 2007).
Several methods have been reported for nitric oxide assay in plants which includes gas chromatography and mass spectrometry (Neil et al., 2003; Conarth et al., 2004, Bethke et al., 2004), laser photo-acoustic spectroscopy (Lesham and Pinchasov, 2000), NO electrode (Yamasaki et al., 2001), electron paramagnetic resonance (EPR) (Sun et al., 2018) and a group of florescent dye indicators which are available in acetylated form for intracellular measurements like Diaminofluorescein-FM (DAF-FM) (Du et al., 2016). Fluorescent dye indicator and EPR both are highly specific for NO. EPR is limited by inability to detect low level NO production and insolubility of chelating agent. Use of fluorogenic probe DAF-FM is gaining more importance because of their simplicity, high sensitivity towards NO and are essentially independent of pH above pH 5.5. This probe is membrane-permeant and deacylated by intracellular esterases to 4-amino-5-methylamino-2 V, 7 V-difluorofluorescein. Presence of light leads to autoxidation of Diaminofluorescein-FM (DAF-FM) dye and simultaneous presence of NO and superoxide source (like xanthine + xanthine oxidase) decreases the fluorescence of Diaminofluorescein-FM (DAF-FM), resulting in underestimation of nitric oxide production (Balcerczyk et al., 2005). This limits the use of Diaminofluorescein-FM (DAF-FM) in stress-related study.
The activity of NOS can be measured by L-citrulline based assay (Tischner et al., 2007) and spectroscopic method (NADPH utilization method) ( Gonzalez et al., 2012). Citrulline-based assay measures the formation of L-citrulline from L-arginine using ion exchange chromatography. The assay does not exactly quantify citrulline; any arginine derivative that does not bind to the cation exchange resin will give a signal and leads to false measurement and also involve radiolabelling which may be tedious to handle (Tischner et al., 2007). Spectrophotometric measurement of NOS activity has been widely regarded as a less expensive and sufficiently sensitive assay for routine laboratorial experiments.
Nitrate reductase activity can be measured by an in vitro or an easy to perform in vivo method (Nair and Abrol, 1973). We have used these protocols to study the effect of elevated CO2 (EC) and nitrate supply on nitrogen metabolism in wheat seedlings (Adavi and Sathee, 2019). S-nitrosylation of NR by EC induced NO produced in plants supplied with high nitrate concentration decreases the enzyme activity (Cheng et al., 2015; Du et al., 2016).
Materials and Reagents
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50 ml glass culture tubes without rim (Borosil, catalog number: 9820U08)
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Scalpel Blade No.10 (Himedia, catalog number: LA76808)
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Paint Brush (Faber-Castell, size-2)
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Glass slide (Himedia, catalog number: LA76808) and cover slip (Himedia, catalog number: GW064)
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Needle (Himedia)
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Kimwipes®
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Moist filter paper
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Butter paper bags
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Wheat seed
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Double distilled water
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2-(4-carboxy phenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (cPTIO, Sigma-Aldrich, 200 µM, catalog number: C221)
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Diaminofluorescein-FM (DAF-FM, Sigma-Aldrich, catalog number: D1821)
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HEPES (Sigma-Aldrich, catalog number: RDD002)
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Potassium nitrate (KNO3) (Fischer Scientific, catalog number:13655)
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N-nitro arginine methyl ester (L-NAME, Sigma-Aldrich, catalog number: N5751)
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Phosphate buffer 200 mM pH 7.5
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Sodium nitroprusside (SNP, Sigma-Aldrich, catalog number: BP453)
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Sodium tungstate (Na-Tungstate, Sigma-Aldrich, catalog number: 14304)
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NEDD (Sisco Research Laboratories Pvt. Ltd, catalog number: 61166)
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N-propanol
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Ammonium Sulfonate (Sisco Research Laboratories Pvt. Ltd, catalog number: 62419)
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HgCl2 (Sisco Research Laboratories Pvt. Ltd, catalog number: 25699)
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Sulfanilamide (Sisco Research Laboratories Pvt. Ltd, catalog number:19689)
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Ferric-EDTA (Sisco Research Laboratories Pvt. Ltd, catalog no 59389)
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Assay buffer (Phosphate buffer, 100 mM, pH 7.0)
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L-Arginine (Sisco Research Laboratories Pvt. Ltd, catalog number: 66637)
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MgCl2 (Sisco Research Laboratories Pvt. Ltd, catalog number: 69396)
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CaCl2 (Sisco Research Laboratories Pvt. Ltd, catalog number: 70650)
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BH4 ((6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride, Sigma-Aldrich, 100 µM, catalog number: 14304)
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FAD (Sisco Research Laboratories Pvt. Ltd, catalog number: 87939)
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FMN (Sisco Research Laboratories Pvt. Ltd, catalog number: 57443)
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DTT (Sisco Research Laboratories Pvt. Ltd, catalog number: 84834)
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PMSF (Sisco Research Laboratories Pvt. Ltd, catalog number: 87606)
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NADPH (Sisco Research Laboratories Pvt. Ltd, catalog number: 77268)
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Bradford reagent (Genetix Biotech Asia Pvt. Ltd, catalog number: E530-1L)
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0.25% HgCl2 in 0.1 N HCl
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7% sulfanilamide in 1 N HCI
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100 µM Sodium nitroprusside (prepared in double distilled water)
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100 µM Sodium tungstate) (prepared in double distilled water)
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0.1% NEDD (prepared in double distilled water)
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0.5% Ammonium sulfonate (prepared in double distilled water)
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0.2 mM NADPH (prepared in double distilled water)
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Nitrogen free Hoagland solution (see Recipes)
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NOS extraction buffer (see Recipes)
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S-nitrosothiol extraction buffer (see Recipes)
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HEPES-KOH with pH 7.5 (see Recipes)
Equipment
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Growth chamber (Conviron, Winnipeg, Canada, model: PGW 36,)
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Watch glass (Himedia, catalog number: LA025)
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Mortar and pestle
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Fluorescence microscope (Zeiss AXIOSKOP 2)
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UV-visible spectrophotometer (Analytik Jena, Germany, model: Specord Bio-200)
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Refrigerated centrifuge (Sigma 3K30)
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Aerator Pump
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Water bath
Software
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ImageJ (https://imagej.nih.gov/ij/download.html)
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MS Excel
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SPSS 10.0
Procedure
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Plant growth
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Pre-soak the wheat seeds in Petri plates lined with moist filter paper till they germinate for 5 days.
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Transfer the uniforms seedlings to 50 ml culture tubes containing nitrogen-free Hoagland solution (Recipe 1). Replenish the Hoagland solution every 3 days (Figure 1). The whole experiment was laid out at National Phytotron Facility, Indian Agricultural Research Institute (IARI), New Delhi in growth chambers (Model PGW 36, Conviron, Winnipeg, Canada).
Growth condition:
Temperature: 25 °C/18 °C (day/night)
Photoperiod: 14 h N/10 h D
Photon flux density: 500 µmol m-2 s-1 (PAR)
Relative humidity (RH): 90%
CO2 concentration: (i) 400 ± 50 µl/l as ambient CO2 (AC)
n(ii) 750 ± 50 µl/l as elevated CO2 (EC)
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Treatments
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After 10 days of transfer, replenish the culture tubes with different set of treatments as mentioned in Table 1. Maintain 3 replication for each treatment.
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Cover the mouth of test tubes with aluminum foil to avoid effect of volatile chemicals on neighbouring plants (Figure 1). Sodium nitroprusside (SNP), a source of NO; cPTIO, an effective NO scavenger-NAME an inhibitor of nitric oxide synthase, and Na-Tungstate, an inhibitor of NR can be used to understand the impact of No on regulation of NR activity. Detailed description on impact of EC on NO localization and NR activity in combination with SNP and inhibitors are discussed in Adavi and Sathee (2019).
Table 1. Treatment details
Figure 1. Representative image of plants grown in 50 ml culture tubes. Each treatment consisted of three tubes with 2 plants per tube.
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Visualization of NO using fluorescent microscope
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After 4 h of treatment, harvest the plants. Cut the root tips into small pieces measuring approximately 2 mm using surgical blade. Then immerse the root sections in the Diaminofluorescein-FM dye (5 µM DAF-FM in 20 mM HEPES-KOH with pH 7.5) in a watch glass for 30 min. After 30 min, carefully take out the root sections and wash with HEPES-KOH buffer without dye 2-3 times till the excess dye is removed.
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Transfer the root sections into glass slide carefully with help of paint brush and cover with cover glass. Drain out excess buffer with help of Kim Wipes.
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Visualize the slides under a fluorescence microscope (Zeiss AXIOSKOP 2) at 495 nm excitation and 515 nm emission wavelengths and acquire the images (Figure 2).
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Analyze the image and calculate the relative fluorescence with “Image J,” a Java-based image processing program.
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Set scale bar on the image using “Image J”.
Figure 2. Representative image of nitric oxide visualized in wheat roots under fluorescent microscope
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Estimation of Nitrate Reductase (NR) activity
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Follow the same procedure for plant growth and treatments as mentioned (Procedures A and B) above. Harvest the plants after 4 h of treatment.
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Estimate in vivo nitrate reductase activity by estimating nitrite formed by the enzyme present in cells and nitrite formed is then diazotized using sulphanilamide in acidic medium and NEDD using the method described by Klepper et al. (1971) and modified by Nair and Abrol (1973). Estimate the nitrite amount using Evans and Nason method (1953).
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Harvest the plants and store in labeled butter paper bags and keep on ice until weighing. Cut the samples (leaves and roots) into 2 mm pieces and mix thoroughly, weigh 0.3 g and add to ice cold incubation medium containing 3 ml each of phosphate buffer (0.2 M, pH 7.5) and potassium nitrate solution (0.4 M). To this, add 0.2 ml of n-propanol.
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Vacuum infiltrate the samples using a pump for 2 min (80-85KPa) and then incubate in a water bath at 30 °C for 30 min under dark conditions.
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After incubation, place the tubes in a water bath (70-80 °C) for 3-4 min to stop the enzyme activity and for the complete leaching of nitrite into the medium.
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Estimate the amount of nitrite produced by taking adequate amount of aliquot (0.2 ml) in a test tube; to it add 1 ml of sulphanilamide (1% in 1 N HCI). After mixing, add 1 ml NEDD (0.02%) and again mix well. Pink color is formed immediately, and after 20 min make the total volume up to 3 ml with double distilled water.
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Measure the absorbance using a UV-visible spectrophotometer (model Specord Bio-200) at 540 nm.
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Prepare the calibration curve using standard potassium nitrite solution.
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Express the enzyme activity as µmol nitrite formed g-1 DW h-1.
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Estimation of Nitric oxide synthase (NOS) activity
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Follow the same procedure for plant growth and treatments as mentioned (Procedures A and B) above. Harvest the plants after 4 h of treatment.
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Homogenize 0.5 g of tissue (leaf and root) samples in 5 ml of cold extraction buffer in pre-chilled mortar and pestle. Centrifuge the homogenate at 10,000 x g for 15 min at 4 °C (Hageman and Hucklesby, 1971). Collect the supernatant to carry out enzyme assays. Determine the protein content of the supernatant by following Bradford’ method (1976).
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Determine the activity of NOS in the reaction mixture (Gonzalez et al., 2012) containing assay buffer (100 mM phosphate buffer pH 7.0), 1 mM L-Arginine, 2 mM MgCl2, 0.3 mM CaCl2, 4 μM BH4, 1 μM FAD, 1 μM FMN, 0.2 mM DTT, 0.2 mM NADPH with 100 μl of tissue extract.
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Observe the change in absorbance due to NADPH utilization at 340 nm for 1 min. Reference was set using reaction mixtures containing distilled water instead of enzyme extract. Three replications of positive control with reaction mixture without L-Arginine was also maintained in each treatment. Use the extinction coefficient of NADPH (ε = 6.22 mM-1 cm-1) for calculating NOS activity.
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Estimation of S-nitrosothiols
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Formation of S-nitrosothiols is estimated from leaf and root tissues (Arc et al., 2013). All the steps need to be performed in dark condition. Follow the same procedure for plant growth and treatments as mentioned (Procedures A and B) above.
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Homogenize 0.5 g of tissue (leaf and root) samples in 5 ml of cold s-nitrosothiols extraction buffer (Recipe 3) in pre-chilled mortar and pestle. Centrifuge the extract at 13,800 x g for 25 min at 4 °C.
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Mix the supernatant with 50 µl ammonium sulfonate and incubate at room temperature for 2 min. Then add 0.3 ml each of sulfanilamide, HgCl2 and NEDD to the reaction mixture. Keep the reaction mixture under dark condition for 20 min at 30 °C.
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Measure the absorbance using a UV-visible spectrophotometer (model Specord Bio-200) at 540 nm.
Data analysis
MS Excel is used for calculations and also to plot the graphs. The least significant difference (LSD at 0.05%) and mean separation using Duncan’s multiple range test was computed by SPSS 10.0. Plants exposed to EC conditions displayed higher accumulation of NO in NOS dependent manner and further details are described in Adavi and Sathee (2019).
Notes
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Care should be taken to avoid exposure of DAF-FM dye to light, as the dye is light sensitive and may lead to auto-oxidation and false fluorescence.
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Culture tube mouth should be properly covered with cotton and aluminium foil to avoid effect of volatile chemicals on neighbouring plants.
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Care should be taken to avoid any damage to root sections while washing or transferring them to slides.
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During measurement of NR, glasswares should be cleaned properly, rinsed with distilled water and air dried to avoid false color development.
Recipes
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Nitrogen free Hoagland Solution
Macronutrient
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Molarity (M)
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Quantity (ml/L of solution)
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K2SO4
|
0.5
|
3.5
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MgSO4·7H2O
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1.0
|
2
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CaCl2
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1.0
|
2
|
KH2PO4
|
1
|
1
|
Micronutrient
|
Quantity (g/L of solution)
|
|
H3BO3
|
2.86
|
|
MnCl2·4H2O
|
1.81
|
|
ZnSO4·7H2O
|
0.22
|
|
CuSO4·5H2O
|
0.08
|
|
Na2MoO4·2H2O
|
0.02
|
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Add micronutrient solution and 0.5% Ferric-EDTA solution (1 ml each) to one liter of solution of macronutrient and adjust the pH to 6.5 prior to use
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NOS extraction buffer
Tris-HCl buffer, 50 mM pH 7.5
10 mM MgCl2
1 mM DTT
1 mM PMSF
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S-nitrosothiol extraction buffer
25 mM HEPES-NaOH
1 mM EDTA, pH 7.8
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HEPES-KOH with pH 7.5
20 mM HEPES was prepared in double distilled water and pH was adjusted to 7.5 with KOH
Acknowledgments
This protocol was adapted from Adavi and Sathee (2019) and our other studies (unpublished). This study was supported by ICAR-Indian Agricultural Research Institute (institute project-CRSCIARISIL20144047279). SAB and BKP acknowledge ICAR- junior research fellowship support received during the course of the study.
Competing interests
The authors declare that there is no conflict of interest.
References
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简介
一氧化氮（NO）是一种氧化还原活性的内源性信号分子，参与许多过程的调节。它在适应和耐受各种非生物和生物胁迫中起着至关重要的作用。在高等植物中，NO是通过酶促还原或非酶促还原亚硝酸盐和需要假定的一氧化氮合酶（NOS）样酶的氧化途径产生的。有几种测量NO产生的方法：质谱法，DAF-FM染料对组织的定位。电子顺磁共振（EPR）也称为电子自旋共振（ESR）和分光光度法。 NOS的活性可以通过基于L-瓜氨酸的测定和光谱法（NADPH利用法）来测定。 NO生物活性转移的主要途径是S-亚硝基化，将NO部分添加到蛋白半胱氨酸硫醇上形成S-亚硝基硫醇（SNO）。该实验方法描述了使用DAF-FM染料通过荧光显微镜（Zeiss AXIOSKOP 2）可视化NO的方法。简化了整个过程，因此易于执行，但对NO检测灵敏度高。此外，还描述了用于测定NOS，硝酸还原酶（NR）和S-亚硝基硫醇含量的基于分光光度法的方案。这些分光光度法操作简便，成本较低且灵敏度足够高，可根据感兴趣的治疗方法提供有关基于NO调节生理过程的足够信息。
【背景】 一氧化氮（NO）逐渐成为多种植物细胞过程的关键调节剂，例如调节细胞壁的合成（Correa-Aragunde et al。，2008; Xiong et al。，2009; Ye et al。，2015），植物中的ROS代谢（Delledonne et al。，2001），基因表达和调控（Bogdan et al。 （，2000），程序性细胞死亡（de Pinto等，2002），成熟和衰老（Yaacov等，1998）。 NO在保护植物免受各种非生物胁迫方面起着至关重要的作用（Hung et al。，2002）。在渗透胁迫下，小麦种子萌发期间，NO可以通过增加过氧化氢酶（CAT），抗坏血酸过氧化物酶（APX）的活性和脯氨酸的积累来显着增强抗氧化能力（Zhang et al。，2003）。 NO生物活性转移的主要途径是S-亚硝基化，将NO部分添加到蛋白半胱氨酸硫醇上形成S-亚硝基硫醇（SNO）。 S-亚硝基化蛋白的总细胞水平主要受S-亚硝基谷胱甘肽还原酶1（GSNOR1）的控制，该酶翻转天然NO供体S-亚硝基谷胱甘肽（GSNO）。在缺少GSNOR1功能的情况下，GSNO会积聚，从而导致总细胞S-亚硝基化的失调（Yun et al。，2016）。
陆地植物一氧化氮的生产通常涉及两条主要途径：第一，还原途径涉及酶和非酶将亚硝酸盐还原为NO（Gupta等，2011）。第二，需要假定的一氧化氮合酶（NOS）样酶的氧化途径。低或无NR活性突变体怀疑NR在NO产生中的作用，该突变体没有可测量的NO。拟南芥的后来的 nia1 / nia2 双突变体证实了NR在将NO 2 -还原为NO中的作用。在体外（Yamasaki等人，，1999）和体内中（Vanin等人），NADH依赖性方式>，2004年）。 NOS可以催化植物中NO合成的可能性也是一个有争议的主要问题。实验证据进一步增加了人们对植物类NOS样酶的怀疑。据报道，通常用于测量植物提取物中一氧化氮合酶活性的基于L-瓜氨酸的测定法容易产生假象（Tischner et al。，2007）。
已经报道了几种用于植物中一氧化氮测定的方法，包括气相色谱法和质谱法（Neil等，2003； Conarth等，2004，Bethke等）。 > et al。，2004年），激光光声光谱法（Lesham and Pinchasov，2000年），NO电极（Yamasaki et al。，2001年），电子顺磁共振（EPR）（ Sun等人，2018年）和一组荧光染料指示剂，它们以乙酰化形式用于细胞内测量，如二氨基荧光素-FM（DAF-FM）（Du等人。 >，2016年）。荧光染料指示剂和EPR均对NO具有高度特异性。 EPR受无法检测低水平NO产生和螯合剂不溶性的限制。荧光探针DAF-FM的使用变得越来越重要，因为它们的简单性，对NO的高敏感性以及基本上与pH 5.5以上的pH无关。该探针是透膜的，并通过细胞内酯酶脱酰基为4-氨基-5-甲基氨基-2V，7V-二氟荧光素。光的存在导致二氨基荧光素-FM（DAF-FM）染料的自氧化，同时存在一氧化氮和超氧化物源（如黄嘌呤+黄嘌呤氧化酶）会降低二氨基荧光素-FM（DAF-FM）的荧光，从而导致一氧化氮的低估生产（Balcerczyk等人，2005年）。这限制了二氨基荧光素-FM（DAF-FM）在压力相关研究中的使用。NOS的活性可以通过基于L-瓜氨酸的测定法（Tischner等，2007）和光谱法（NADPH利用法）进行测定（Gonzalez等，2012）。 ）。基于瓜氨酸的测定法使用离子交换色谱法测量了从L-精氨酸形成L-瓜氨酸的过程。该测定法不能准确地定量瓜氨酸。任何不与阳离子交换树脂结合的精氨酸衍生物都会发出信号并导致错误的测量结果，并且还涉及放射性标记，可能难以处理（Tischner et al。，2007）。对于常规实验室实验，NOS活性的分光光度测量已被广泛认为是一种较便宜且足够灵敏的测定方法。
硝酸还原酶的活性可以通过体外或易于实施的体内方法进行测量（Nair and Abrol，1973）。我们已使用这些协议研究了CO 2 （EC）和硝酸盐供应升高对小麦幼苗氮代谢的影响（Adavi和Sathee，2019）。硝酸盐浓度高的植物中EC诱导的NO引起的NR的S-亚硝基化会降低酶的活性（Cheng et al。，2015; Du et al。，，2016） 。
关键字：一氧化氮, 一氧化氮合酶（NOS）样蛋白, 硝酸还原酶（NR）, 二氨基荧光素-FM（DAF-FM）, S-亚硝基硫醇