Materials and Reagents

50 mL centrifuge tube with round bottom
2 mL centrifuge tube with round bottom
70 μm Nylon mesh (EMD Millipore, CLS431751)
3–4 weeks old Arabidopsis plants
Scintillation fluid (Perkin-Elmer, Waltham, catalog number: 6013321)
¹⁴C-paraquat (American Radiolabeled Chemicals Inc. ARC 1311). Store at -20 °C
NaCl (Sinopharm Chemical Reagent Co., Ltd., CHINA, CAS: 7647-14-5)
CaCl₂(Sinopharm Chemical Reagent Co., Ltd., CHINA, CAS: 10043-52-4)
KCl (Sinopharm Chemical Reagent Co., Ltd., CHINA, CAS: 7447-40-7)
MES (SIGMA, USA CAS: 1266615-59-1)
W5 solution (see Recipes)

Equipment

Centrifuge with temperature control (Allegra X-22R Benchtop Centrifuge, Beckman Coulter, Indianapolis, USA)
Automated cell counter (IC1000, Shanghai RuiYu Biotech Co. Ltd., Shanghai, CHINA)
Ventilation cabinet (YKD-DAT004F, Wuxi Safoo Safety Equipment Co. Ltd., Wuxi, CHINA)
Scintillation vial (Perkin-Elmer, Waltham, USA)
Liquid scintillation counter (Tri-Carb 2910 TR, Perkin-Elmer, Waltham, USA)
-20 °C freezer

Procedure

Isolate protoplasts according to Yoo et al. (2007) and suspend in cold W5 solution (see Recipes).
Count the protoplasts with a cell counter to estimate their concentration. Centrifuge at 40 × g and 4 °C for 2 min. Decant the supernatant and resuspend the protoplasts in cold W5 solution to approximately 3 × 10⁶ protoplasts/mL. This protoplast preparation is ready for ¹⁴C-paraquat loading (Figure 1).

Figure 1. Experimental scheme of ¹⁴C-paraquat loading into and efflux out of Arabidopsis mesophyll protoplasts.
Thaw out ¹⁴C-paraquat (3.1 mM, 0.1 μCi/μL) and dilute 10 times with W5 solution at room temperature.
Add the same volume of diluted ¹⁴C-paraquat solution to the protoplast preparation, to obtain a final concentration of 3 μM ¹⁴C-paraquat. Incubate for 1 h at room temperature to load paraquat into the protoplasts.
Centrifuge the protoplasts at 40 × g and 4 °C for 2 min. Discard the supernatant with the unloaded ¹⁴C-paraquat and wash the protoplasts with 15 mL of cold W5 solution 2–3 times.
Resuspend the protoplasts in 13 mL of W5 solution at room temperature and immediately initiate the paraquat efflux assay.
At different time points (0, 10, 20, and 40 min) after W5 solution resuspension, transfer 1 mL of protoplasts suspension into a 2 mL round-bottom centrifuge tube. Take at least three replicates at each time point.
Immediately centrifuge the aliquoted samples (40 × g, 2 min, 4 °C). Transfer the supernatant to a scintillation vial. Resuspend the protoplast pellet in 1 mL of W5 solution and transfer it to a scintillation vial. Then add 5 mL of scintillation fluid to each sample. Detect radioactivity on a liquid scintillation counter.
Analyze and present data as shown in Figure 2.

Note: ¹⁴C-paraquat waste was disposed according to the University’s safety management of isotopes.

Figure 2. Data analyses and presentation. (A) Calculation of ¹⁴C-paraquat retention in protoplasts at different time points. t=i: time of ¹⁴C-paraquat protoplasts efflux (0, 10, 20, and 40 min). CPM_t=i: mean value of ¹⁴C-paraquat radioactivity retention in protoplasts at different time points. CPM_t=0: mean value of ¹⁴C-paraquat radioactivity retention in protoplasts at the initial time. A_t=i: percentage of ¹⁴C-paraquat radioactivity retention in protoplasts at different time points relative to the initial time. (B) Paraquat retention in the protoplasts, measured in the Col-0 and DTX6 overexpression line (OE-13) (Xia et al., 2021). Paraquat retention was defined as a percentage of the ¹⁴C-paraquat loaded in the protoplasts. Values are mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05; one-way ANOVA).

Recipes

W5 solution
154 mM NaCl
125 mM CaCl₂
5 mM KCl
2 mM MES
pH 5.7

Acknowledgments

This protocol was adapted from previous work (Zhang et al., 2014; Qin et al., 2021); we would like to thank the authors of these studies for their protocols.

Competing interests

No conflict of interest declared.

References

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