Determination of Paraquat in Arabidopsis Tissues and Protoplasts by UHPLC-MS/MS

Materials and Reagents

1. 50 mL conical centrifuge tube (PROMETHE, catalog number: PCT010500, sterile)

2. 15 mL conical centrifuge tube (PROMETHE, catalog number: PCT010150, sterile)

3. 2 mL centrifuge tube (Shanghai Jing Xin, catalog number: JX-LG0100, nonsterile)

4. 2.0 mL amber screw vial (Thermo Fisher, catalog number: C5000-2W)

5. 150 μL liner pipe (Thermo Fisher, catalog number: C4012-530)

6. Two-week-old Arabidopsis seedlings grown on 1/2 MS plates (mutant seeds are available via email request to the corresponding author, and wildtype seeds are available from TAIR website https://www.arabidopsis.org)

7. Four-week-old Arabidopsis plants grown in soil

8. Murashige & Skoog basal medium with vitamins (PhytoTech, catalog number: M519)

9. KCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7447-40-7)

10. CaCl₂ (Solarbio, CAS: 10043-52-4)

11. NaCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7647-14-5)

12. HCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7647-01-0)

13. MES (BBI, CAS: 145224-94-8)

14. NH₄OAC (Sinopharm Chemical Reagent Co., Ltd, CAS: 631-61-8)

15. Paraquat (Aladdin, CAS: 1910-42-5)

16. Water (LC-MS-grade) (Merck, Milli-Q IQ7000)

17. Methanol (LC-MS-grade) (Merck, CAS: 67-56-1)

18. Acetonitrile (LC-MS-grade) (Sigma-Aldrich, CAS: 75-05-8)

19. Formic acid (LC-MS-grade) (Sigma-Aldrich, CAS: 64-18-6)

20. Liquid nitrogen

21. W5 buffer (see Recipes)

22. Paraquat treatment buffer (see Recipes)

23. Wash buffer (see Recipes)

24. Paraquat extraction solution (see Recipes)

25. Redissolving solution (see Recipes)

26. LC mobile phase (see Recipes)

Equipment

1. Automatic sample fast grinder (Shanghai Jing Xin, model: JXFSTPRP-CL)

2. Ultrasonic cleaners (Supmile, model: KQ-200VDE)

3. Nitrogen blowing concentrator (Beijing Jiayuan Industrial Technology Co., Ltd., model: MD200-2)

4. High-speed refrigerated centrifuge (KUBOTA, model: 6500)

5. Table high-speed centrifuge (Hettich, model: MIKRO 220R)

6. Light microscopy (Leica, model: Stereo microscope S8 APO)

7. -80 freezer

8. Water bath (HEDE Laboratory, model: DK-420BS)

9. Hemocytometer

10. UHPLC system (Shimadzu Nexera UHPLC LC30A system, including sample manager, solvent manager, and column oven)

11. Mass spectrometer system (SCIEX QTRAP 6500 plus mass spectrometer equipped with an electrospray ionization)

12. UHPLC column (ACQUITY UPLC BEH HILIC Column, 130Å, 1.7 µm particle size, 2.1 mm × 100 mm, Waters)

Procedure

1. Treat Arabidopsis seedlings with paraquat

   1. Surface-sterilize the Arabidopsis seeds with 70% ethanol and then grow them on vertical 1/2 Murashige & Skoog square plates (1% agar) for 10 days under standard long daylight growth conditions (16:8 h light/dark cycle) (Weigel and Glazebrook, 2002).

   2. Lay the plates horizontally and then leave them in darkness for two days to allow the hypocotyl to grow upward quickly. After that, return the plates to long daylight conditions again to grow for another two days (Figure 1).

      
      
      Figure 1. Brief diagram of seedling growth and treatment process

      
      

      Note: On each plate, mutants and the Col-0 wildtype control were grown side-by-side. The growth procedure illustrated in Figure 1 will facilitate the subsequent paraquat treatment and root harvest. Roots mainly grow on the surface of the media and can easily be treated and harvested, while leaves do not touch the media surface.

   3. Use a pipette to add 10 mL of 10 μM paraquat slowly into the plates and let the paraquat distribute evenly on the media surface. Keep the plates horizontally in darkness for 6 h.

      Note: Use the same quantity of paraquat to treat each plate. The volume of paraquat should be enough to submerge the media surface but not enough to touch the leaves.

   4. Harvest roots and leaves separately. Rinse the materials shortly three times with distilled water, blot them dry with paper towels, and then weigh. For mutants and controls, it is better to use a similar weight of materials for further experiments.

   5. Freeze the materials in liquid nitrogen and store them at -80 °C for paraquat determination.




2. Treat Arabidopsis protoplasts with paraquat

   1. Prepare protoplasts from the leaves of Arabidopsis grown under short daylight conditions (8:16 h light/dark cycle) for four weeks based on the method described previously (Yoo et al., 2007). Suspend the protoplasts in cold W5 buffer.

   2. Count the protoplasts using a hemocytometer. Remove the W5 buffer and adjust the protoplasts to a final concentration of 2 × 10⁶ protoplasts/mL with paraquat treatment solution. Use at least 10 mL protoplasts for one assay.

   3. Incubate the protoplasts in darkness at room temperature for 3 h. Gently shake the protoplasts by hand every 20 min.

      Note: This step is to load the paraquat into the protoplasts.

   4. Centrifuge at 100 × g for 2 min at 4 °C and remove the supernatant carefully.

   5. Add 10 mL of wash buffer into the tube and resuspend the protoplasts gently.

   6. Repeat steps B4–B5 two more times.

      Note: The above steps can be used for paraquat uptake assay. The following steps are carried out for paraquat efflux assay.

   7. After the final wash, suspend the protoplasts in wash buffer again and aliquot them equally into several tubes. Each tube corresponds to one time point. Set at least three replicates for each time point. For this assay, we use 0, 10, 30, and 60 min time points. Keep the tubes in darkness at room temperature and start timing using a timer.

   8. At each time point, centrifuge the tubes as in step B4. Collect the supernatants and protoplasts, respectively.

   9. Freeze the samples in liquid nitrogen and then store them at -80 °C for paraquat determination.




3. Paraquat extraction

   1. Use 1 × 10⁶–2 × 10⁶ protoplasts or 20–30 mg of plant tissues to determine paraquat content in a 2 mL centrifuge tube. Conduct three freeze-thaw cycles (freeze the protoplasts in liquid nitrogen for 1 min, then thaw them in a water bath at room temperature for 2 min) to rupture the protoplasts. For plant tissues, grind the materials into powder in liquid nitrogen.

   2. Add 800 µL of paraquat extraction solution into each tube.

   3. Sonicate each sample for 20 min (1 min on, 1 min off) in total on ice.

   4. Put the samples in a water bath at 60 °C for 30 min and then cool down to room temperature.

   5. Centrifuge at 13,500 × g for 10 min at 4 °C and collect the supernatant.

   6. Repeat steps C2–C5 and combine the supernatants together.

   7. Evaporate the organic solvents with a nitrogen blowing concentrator and store the dried samples at -80 °C until determination.




4. Paraquat determination

   1. Take the samples from -80 °C and add 100 μL of the room-temperature redissolving solution into the 2 mL centrifuge tubes to dissolve the samples.

   2. Lightly flick the tubes several times until the samples are completely dissolved. Centrifuge at 14,000 × g for 10 min at 4 °C. Pipette 80 μL of the supernatant into a 2 mL amber screw vial with a 150 μL liner pipe.

   3. Inject 1 μL of the sample into the system with a Waters BEH HILIC column (1.7 μm particle size, 2.1 mm × 100 mm).

   4. Perform a linear gradient elution program using LC mobile phase consisting of solvent A and solvent B. Set the flow rate of the mobile phase at 0.4 mL/min to conduct a linear gradient elution program (7 min): 90% B hold for 1 min, 90% B decreased to 20% B in 1 min, 20% B hold for 2.3 min, 20% B increased to 90% B in 0.2 min, and 90% B hold for 2.5 min. (The procedure was 0 min, 90% B; 1 min, 90% B; 2 min, 20% B; 4.3 min, 20% B; 4.5 min, 90% B; and 7 min, 90% B.)

   5. Detect the samples in positive mode (ESI+) with multiple reaction monitoring mode on the LC-MS/MS system.

      Set the gas parameters in ion source, including ion source gas 1, ion source gas 2, and the curtain gas to 55, 55, and 35 psi, respectively. Set the temperature to 500 °C and the ion spray voltage to 5,500 V. Monitor two multiple reaction transitions of each analyte for quantification and confirmation of paraquat (186.0–171.0 for quantification and 93.2–171.3 for confirmation). The voltages of declustering potential and collision energy are compound-specific parameters. For paraquat, they are 100, 60, 25, and 17, respectively. Set other parameters to default values as recommended by the manufacturer.

   6. Integrate the chromatographic peak of the target analyte. Integration parameters were set to 9 and 1,000 points for the minimum peak width and height, and S/N integration threshold and baseline subtraction window were set to 3 and 2 min, respectively.

Data analysis

1. Calculate the chromatographic peak area of paraquat directly by UHPLC-MS/MS. The peak area of paraquat represents the amount of analyte on the column. Since the injection volume is 1 µL and the total volume of the sample is 100 µL, we multiply the chromatographic peak area by 100 to obtain the total quantity of analyte in the sample.

2. Paraquat efflux assay results are presented in Figure 2A. The same number of protoplasts was sampled at each time point for paraquat determination. Paraquat content in protoplasts was indicated by its chromatographic peak area. The paraquat level at time point 0 was set as 100% and the relative levels of paraquat at other time points were calculated according to the formula in Figure 2B.

3. Root uptake assay results are presented in Figure 2C. The normalized paraquat level in tissues was obtained by dividing the chromatographic peak area of paraquat by the quantity of materials, that is, the weight of Arabidopsis materials (such as roots and leaves). The paraquat content in different samples was compared.

   
   

   
   Figure 2. Paraquat determination in protoplasts and plant tissues by UHPLC-MS/MS. (A) Paraquat efflux assay using Arabidopsis leaf protoplasts. The paraquat level in protoplasts was indicated by its chromatographic peak area. At least three replicates for each time point were determined. The paraquat level at time point 0 was set as 100%, and the relative levels of paraquat at other time points were calculated using the formula given in Figure 2(B). The samples shown are Col-0 (wildtype control), DTX6m-11 (DTX6m-overexpression line), and dm-5 (dtx5 dtx6 double mutant) (Lv et al., 2021). Values are mean ± SD (n = 3). Significance of the differences between Col-0 and the other materials is analyzed by Student’s t-test. PQ, paraquat. ** P ≤ 0.01; * P ≤ 0.05. (B) Calculation formula of relative paraquat level at different time points in Arabidopsis protoplasts. PA_t: peak area of paraquat at each time point; PA₀: peak area of paraquat at time point 0. (C) Paraquat uptake assay using Arabidopsis roots. Arabidopsis were treated with 10 μM paraquat in darkness for 6 h and the paraquat level in tissues was defined as the chromatographic peak area of paraquat divided by the fresh weight of the tissues. Values are mean ± SD (n = 3). Two-way ANOVA is performed to show the significance of difference.

Recipes

1. W5 buffer (for protoplast experiment)

   154 mM NaCl

   125 mM CaCl₂

   5 mM KCl

   2 mM MES (pH 5.7)

   Note: Prepare mother solutions for each reagent, then filter and store them at 4 °C. Make W5 buffer freshly using mother solutions.




2. Paraquat treatment solution (for protoplast experiment)

   154 mM NaCl

   125 mM CaCl₂

   5 mM KCl

   2 mM MES (pH 5.7)

   5 mM paraquat

   Note: Make this solution freshly using the stored mother solutions of each component. The mother solution of paraquat should be stored at -20 °C in the dark.




3. Wash buffer (for protoplast experiment)

   154 mM NaCl

   125 mM CaCl₂

   5 mM KCl

   2 mM MES (pH 5.7)

   Note: Make wash buffer freshly using the stored mother solutions.




4. Paraquat extraction solution

   60% methanol

   40% 0.5 M HCl

   Note: This solution can be stored at -20 °C for three months.




5. Redissolving solution

   50% acetonitrile

   50% Milli-Q water

   Note: Make this solution freshly.




6. LC mobile phase

   Solvent A (pH = 3.95):

   100 mM NH₄OAC

   0.5% formic acid in water

   Solvent B:

   Acetonitrile

   Note: Make solvent A freshly.

Acknowledgments

This protocol was developed based on the methods described previously (Pizzutti et al., 2016; Zou et al., 2015). This work was supported by the National Natural Science Foundation of China (Grants No. 31970343 and 31770274)

Competing interests

No conflict of interest declared.

References

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