Sulfatase Assay to Determine Influence of Plants on Microbial Activity in Soil

Materials and Reagents

1. Tape
2. 0.5 ml Eppendorf tubes
   
3. Toothpicks
4. 1.5 ml cuvettes
5. Plastic trays, Plant Pots Direct, Heavyweight full seed tray (no holes), catalog number: 2012137PT (Figure 1)
6. Plastic inserts (Plant Pots Direct, Seed Tray Inserts 40, catalog number: 2012111PT) (Figure 1)
7. Plastic Petri dishes (Sarstedt, catalog number: 821.473)
8. Pipette tips (Sarstedt)
9. 2 ml plastic tubes (Sarstedt, catalog number: 72.695.500)
10. Plastic rack for 2 ml tubes, Eppendorf
11. Plastic cuvettes (Brand, catalog number: 759115)
12. Seeds of Arabidopsis thaliana (can be obtained, e.g., from NASC Arabidopsis Stock Centre, http://arabidopsis.info/BasicForm)
13. Sand, Quarzwerke (Frechen, Germany)
14. Soil (e.g., CAS11 soil, Bulgarelli et al., 2012)
15. Murashige Skoog (MS) medium (Duchefa Biochemie, catalog number: MO222.0025), stored at RT
16. Sucrose (Sigma, catalog number: S7903-1KG), stored at RT
17. Potassium-4-nitrophenyl sulfate (Sigma, catalog number: N3877-1G), stored at -20 °C
18. P-nitrophenol (Merck, catalog number: 48549), stored at -20 °C
19. Calcium chloride dihydrate (Sigma, catalog number C3881-1KG), stored at RT
20. Sodium hydroxide (NaOH) (Sigma, catalog number: 71687-500G), stored at RT
21. Sodium acetate trihydrate (Sigma, catalog number: S8625-500G), stored at RT
22. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) (Merck, catalog number: C1396), stored at RT
23. Potassium nitrate (KNO₃) (Merck catalog number: P8394), stored at RT
24. Potassium dihydrogenphosphate (KH₂PO₄) (Merck, catalog number: 1.04873), stored at RT
25. Ferric EDTA (Fe-EDTA) (Merck, catalogue number: E6760), stored at RT
26. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Merck, catalog number: M2670), stored at RT
27. Liquid nitrogen
28. Glacial acetic acid (Merck, catalog number: A6283), stored at RT
29. Sodium hypochlorite solution (NaClO), 12%, Cl (Roth, catalog number: 9062.4)
30. Hydrochloric acid (HCl) 37% (Merck KGaA, catalog number: 1.00317.1000), stored at RT
31. Agarose (Sigma, catalog number: A9539-500G), stored at RT
32. Toluene (Sigma, catalog number: 34866-100 ml), stored under fume hood at RT
33. Modified Long Ashton solution (see Recipes)
34. 0.5 M Acetate buffer (see Recipes)
35. 0.005 M p-nitrophenyl solution (see Recipes)
36. 0.5 M calcium chloride solution (see Recipes)
37. 0.5 M sodium hydroxide solution (see Recipes) 
38. Standard p-nitrophenol solution 10 mM (100 ml) (see Recipes)
39. Half strength MS medium with sucrose (1 L) (see Recipes)
    
    
    Figure 1. Photos of (A) plastic trays and (B) inserts used for plant growth
    

Equipment

1. Glass beaker 250 ml
2. Forceps
3. Pipettes (Eppendorf)
4. Balances (Sartorius)
5. Glass beaker
6. Sanyo growth chamber, 10 h light/14 h darkness, 22 °C
7. Rotating shaker, LTF (Labortechnik, Intelli-Mixer, RM-2L)
8. Vortex (LMS, model: VTX-3000L)
9. Incubator for 37 °C (Thermo Scientific, Heratherm Incubator)
10. Centrifuge (Eppendorf, 5424)
11. Spectrophotometer (Eppendorf)
12. Fume hood
13. Computer
14. Desiccator
    

Procedure

1. Arabidopsis thaliana seed sterilization
   1. Place small portion of seeds (ca. 10 µl) into 0.5 ml Eppendorf tubes.
   2. Place open tubes into tube rack inside of desiccator (Figure 2).
   3. Add 125 ml of sodium hypochlorite solution into 250 ml glass beaker and place near the seeds.
   4. Add 2.5 ml of concentrated HCl into the liquid, which forms chlorine gas, and quickly close the desiccator lid.
   5. Sterilize seeds for 3 h.
      
      
      Figure 2. Photos of (A) open or (B) closed desiccator for seed sterilization
      
      
      
2. Initial plates preparation
   1. Autoclave half-strength MS medium with 0.8% agarose and 0.5% sucrose and pour it into round Petri dishes, let it set.
   2. Carefully place sterile Arabidopsis seeds onto agar, using sterile toothpicks, approx. 1 seed per cm².
   3. Seal the plates with tape.
   4. Place the plates into fridge for 2-3 days for stratification.
   5. Place plates into a plant growth cabinet for 9 days (10 h light/14 h dark; 22 °C, 100 µE m^-2 s^-1).
      
      
      
3. Preparation of trays
   1. Mix soil with sterile (autoclaved) sand 1:9 (V/V).
   2. Place plastic insert into the tray.
   3. Fill inserts with soil-sand mix.
   4. Water slightly.
   5. Using forceps carefully transfer one seedling per insert, covering roots lightly.
   6. Water a bit more, to keep soil moist.
   7. Cover tray with the lid.
   8. Place trays into plant growth cabinet (10 h light/14 h dark; 22 °C, 100 µE m^-2 s^-1) for 2 weeks, uncover it after 3 days, and water daily with modified Long Ashton solution.
      
      
      Figure 3. Photos of (A) Petri dish with seedlings before transfer and (B) trays with Arabidopsis ecotypes ready for material collection
      
      
      
4. Collection of samples for sulfatase activity
   1. Carefully remove plants from soil.
   2. Into a 2 ml microcentrifuge tube collect about 1 g of the soil-sand mix, which was closest to the roots of the plant, i.e., rhizosphere, record fresh weight.
   3. Collect at least 2 samples per plant.
   4. Freeze in liquid nitrogen.
      
      
      
5. Sulfatase activity measurement
   1. Defrost the samples in a rack.
   2. Add 400 µl of 0.5 M acetate buffer.
   3. Vortex each sample for at least 5 s.
   4. Under fume hood add 25 µl of toluene.
   5. Close tubes and vortex for at least 5 s.
   6. Place tubes into rotating rack for 6 min at 100 rpm.
   7. Add 100 µl of p-nitrophenyl sulfate solution under fume hood.
   8. Vortex each sample for 10 s.
   9. Place tubes for additional vigorous shaking for 5 min in an Eppendorf shaker at 1,000 rpm.
   10. Place rack with the tubes into 37 °C incubator for 1 h, mixing the whole rack every 10 min by reversing several times.
   11. To stop the reaction under fume hood add 100 µl of 0.5 M CaCl₂ solution and 400 µl of 0.5 M sodium hydroxide solution.
   12. Vortex each sample for 10 s.
   13. Centrifuge tubes at RT at maximum speed for 20 min.
   14. Under fume hood carefully transfer the supernatant into plastic 1.5 ml cuvettes.
   15. Measure absorption at 400 nm. Use water as blank.
   16. Prepare standards by diluting 0, 20, 40, 80, 120, 160, and 200 µl of 10 mM p-nitrophenol standard to 1 ml H₂O and measure in the same way.
   17. Calculate the p-nitrophenol content of the samples from a calibration curve (standards: 0, 200, 400, 800, 1,200, 1,600, and 2,000 nmol ml^-1 p-nitrophenol).
   18. Using recorded FW data and incubation time of 1 h calculate sulfatase activity in nmol g FW^-1 h^-1.

Data analysis

The assay determines the end concentration of the sulfatase product, p-nitrophenol, using a calibration curve (Figure 1) and the activity can be calculated from the weight of soil and time of the assay. All calculations can be easily performed in standard office software, e.g., Excel (Table 1). Five biological replicates with two technical replicates each should be used for each plant genotype.


Figure 4. Calibration curve for measurement of sulfatase activity. Standard p-nitrophenol solution was diluted by water to contain 0-2,000 nmol per ml and OD₄₀₀ was measured.

Table 1. Example of calculation of sulfatase activity

¹The soil FW is adjusted from the FW of soil/sand sample and the ratio of soil/sand mix, here 10% soil.

Notes

The assay can easily be adapted for different plant species, different sizes of pots, or different soils.

Recipes

1. Modified Long Ashton solution
   1.5 mM Ca(NO₃)₂·4H₂O
   1 mM KNO₃
   0.75 mM KH₂PO₄
   0.1 mM Fe-EDTA
   0.75 mM MgCl₂·6H₂O
   pH 5.7
2. 0.5 M acetate buffer, pH 5.8 (1 L)
   64 g sodium acetate trihydrate
   1.7 ml glacial acetic acid
   Stored at 4 °C
3. 0.005 M p-nitrophenyl sulfate solution (100 ml)
   0.1287 g of p-nitrophenyl dissolved in 100 ml of 0.5 M acetate buffer
   Stored at 4 °C
4. 0.5 M calcium chloride solution (1 L)
   73.5 g CaCl₂·2H₂O dissolved in 1 L H₂O
   Stored at RT
5. 0.5 M sodium hydroxide solution (1 L)
   20 g NaOH dissolved in 1 L H₂O
   Stored at RT
6. Standard p-nitrophenol solution 10 mM (100 ml)
   139 mg p-nitrophenol dissolved in 100 ml H₂O
   Stored at 4 °C
7. Half strength MS medium with sucrose (1 L)
   2.2 g MS medium
   5 g sucrose
   8 g agarose
   pH 5.7 with 1 M KOH
   

Acknowledgments

The sulfatase assay was adapted from Margesin et al., 2014. SK’s research is funded by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy, EXC-Nummer 2048/1, project 390686111. AS was funded by FP7-Peoople (293429) and the Irish Research Council New Foundation “S-cycle”.

Competing interests

The authors declare no competing interests.

References

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