Materials and Reagents

50 ml glass culture tubes without rim (Borosil, catalog number: 9820U08)
Scalpel Blade No.10 (Himedia, catalog number: LA76808)
Paint Brush (Faber-Castell, size-2)
Glass slide (Himedia, catalog number: LA76808) and cover slip (Himedia, catalog number: GW064)
Needle (Himedia)
Kimwipes^®
Moist filter paper
Butter paper bags
Wheat seed
Double distilled water
2-(4-carboxy phenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (cPTIO, Sigma-Aldrich, 200 µM, catalog number: C221)
Diaminofluorescein-FM (DAF-FM, Sigma-Aldrich, catalog number: D1821)
HEPES (Sigma-Aldrich, catalog number: RDD002)
Potassium nitrate (KNO₃) (Fischer Scientific, catalog number:13655)
N-nitro arginine methyl ester (L-NAME, Sigma-Aldrich, catalog number: N5751)
Phosphate buffer 200 mM pH 7.5
Sodium nitroprusside (SNP, Sigma-Aldrich, catalog number: BP453)
Sodium tungstate (Na-Tungstate, Sigma-Aldrich, catalog number: 14304)
NEDD (Sisco Research Laboratories Pvt. Ltd, catalog number: 61166)
N-propanol
Ammonium Sulfonate (Sisco Research Laboratories Pvt. Ltd, catalog number: 62419)
HgCl₂ (Sisco Research Laboratories Pvt. Ltd, catalog number: 25699)
Sulfanilamide (Sisco Research Laboratories Pvt. Ltd, catalog number:19689)
Ferric-EDTA (Sisco Research Laboratories Pvt. Ltd, catalog no 59389)
Assay buffer (Phosphate buffer, 100 mM, pH 7.0)
L-Arginine (Sisco Research Laboratories Pvt. Ltd, catalog number: 66637)
MgCl₂ (Sisco Research Laboratories Pvt. Ltd, catalog number: 69396)
CaCl₂ (Sisco Research Laboratories Pvt. Ltd, catalog number: 70650)
BH₄ ((6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride, Sigma-Aldrich, 100 µM, catalog number: 14304)
FAD (Sisco Research Laboratories Pvt. Ltd, catalog number: 87939)
FMN (Sisco Research Laboratories Pvt. Ltd, catalog number: 57443)
DTT (Sisco Research Laboratories Pvt. Ltd, catalog number: 84834)
PMSF (Sisco Research Laboratories Pvt. Ltd, catalog number: 87606)
NADPH (Sisco Research Laboratories Pvt. Ltd, catalog number: 77268)
Bradford reagent (Genetix Biotech Asia Pvt. Ltd, catalog number: E530-1L)
0.25% HgCl₂ in 0.1 N HCl
7% sulfanilamide in 1 N HCI
100 µM Sodium nitroprusside (prepared in double distilled water)
100 µM Sodium tungstate) (prepared in double distilled water)
0.1% NEDD (prepared in double distilled water)
0.5% Ammonium sulfonate (prepared in double distilled water)
0.2 mM NADPH (prepared in double distilled water)
Nitrogen free Hoagland solution (see Recipes)
NOS extraction buffer (see Recipes)
S-nitrosothiol extraction buffer (see Recipes)
HEPES-KOH with pH 7.5 (see Recipes)

Equipment

Growth chamber (Conviron, Winnipeg, Canada, model: PGW 36,)
Watch glass (Himedia, catalog number: LA025)
Mortar and pestle
Fluorescence microscope (Zeiss AXIOSKOP 2)
UV-visible spectrophotometer (Analytik Jena, Germany, model: Specord Bio-200)
Refrigerated centrifuge (Sigma 3K30)
Aerator Pump
Water bath

Software

ImageJ (https://imagej.nih.gov/ij/download.html)
MS Excel
SPSS 10.0

Procedure

Plant growth
1. Pre-soak the wheat seeds in Petri plates lined with moist filter paper till they germinate for 5 days.
2. 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)
Treatments
1. 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.
2. 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.
Visualization of NO using fluorescent microscope
1. 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.
2. 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.
3. 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).
4. Analyze the image and calculate the relative fluorescence with “Image J,” a Java-based image processing program.
5. Set scale bar on the image using “Image J”.
  
  Figure 2. Representative image of nitric oxide visualized in wheat roots under fluorescent microscope
Estimation of Nitrate Reductase (NR) activity
1. Follow the same procedure for plant growth and treatments as mentioned (Procedures A and B) above. Harvest the plants after 4 h of treatment.
2. 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).
3. 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.
4. 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.
5. 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.
6. 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.
7. Measure the absorbance using a UV-visible spectrophotometer (model Specord Bio-200) at 540 nm.
8. Prepare the calibration curve using standard potassium nitrite solution.
9. Express the enzyme activity as µmol nitrite formed g^-1 DW h^-1.
Estimation of Nitric oxide synthase (NOS) activity
1. Follow the same procedure for plant growth and treatments as mentioned (Procedures A and B) above. Harvest the plants after 4 h of treatment.
2. 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).
3. 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.
4. 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.
Estimation of S-nitrosothiols
1. 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.
2. 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.
3. 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.
4. 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

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.
Culture tube mouth should be properly covered with cotton and aluminium foil to avoid effect of volatile chemicals on neighbouring plants.
Care should be taken to avoid any damage to root sections while washing or transferring them to slides.
During measurement of NR, glasswares should be cleaned properly, rinsed with distilled water and air dried to avoid false color development.

Recipes

Nitrogen free Hoagland Solution

Macronutrient	Molarity (M)	Quantity (ml/L of solution)
K₂SO₄	0.5	3.5
MgSO₄·7H₂O	1.0	2
CaCl₂	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

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

NOS extraction buffer
Tris-HCl buffer, 50 mM pH 7.5
10 mM MgCl₂
1 mM DTT
1 mM PMSF
S-nitrosothiol extraction buffer
25 mM HEPES-NaOH
1 mM EDTA, pH 7.8
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.

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