Materials and Reagents

3-week old Arabidopsis plants
Mannitol (BDH Prolabo, catalog number: 25311 )
CaCl₂ (POCH, catalog number: M00015143 )
KCl (USB, catalog number: 20598 )
2-(N-morpholino)ethanesulfonic acid (MES) (LabEmpire, catalog number: MES503 )
NaCl (POCH, catalog number: BA4121116 )
Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 81240 )
Cellulase (SERVA Electrophoresis GmbH, catalog number: 16419 )
Macerozyme (SERVA Electrophoresis GmbH, catalog number: 28302 )
GenElute^TM HP Plasmid Midiprep Kit (Sigma-Aldrich, catalog number: PLD35 )
Benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal, Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich, catalog number: M7449 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
MgCl₂ (USB, catalog number: 18641 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Protease inhibitors (Roche Diagnostics, catalog number: 11873580001 )
Enzyme solution (see Recipes)
W5 (see Recipes)
W1 (see Recipes)
MMg (see Recipes)
40% PEG (see Recipes)
Protein isolation buffer (see Recipes)

Equipment

Light microscope (Nikon Corporation)
Hemocythometer (Sigma-Aldrich)
Tape (Scotch^® Magic^TM)
Stainless steel forceps (Sigma-Aldrich)
Scissors
10 ml pipette
50 ml tubes
90 mm Petri dishes
Amicon Ultra-15 centrifugal filter unit (Millipore)
NanoDrop spectrophotometer
Tabletop centrifuge
Horizontal shaker

Procedure

Protoplast isolation
1. Using tweezers, 5-6 mature leaves of Arabidopsis, petioles removed, were harvested.
2. Adaxial surface of leaves was put on a piece of tape and leaves were flattened (Figure 1A-B).
3. Second piece of tape was applied with limited pressure, trapping leaves between them (Figure 1C). To much pressure will result in damaging leaves, too little pressure will lead to poor epidermis removal effectiveness.
4. Sandwich was placed with adaxial surface on top and pieces of tape were split by pulling away tape on top side, started at the tip of leaf (Figure 1D-E) (see Note 2).
  
  Figure 1. Preparation of Arabidopsis leaf for protoplast isolation. A. 3-weeks old Aradodopsis thaliana; B-E. Step by step procedure of leaf epidermis removal with tape.
5. Tape was cut around leaves and put at room temperature enzyme solution in Petri dish, exposed mesophyll down. Tape should not be immersed in solution, but float on top of it. Mesophyll cells were digested for 60 min at 30 °C with gentle shaking (55 rpm) (see Note 3).
6. After digestion pieces of tape were removed and cells were left for another 5 min in same conditions (see Note 4).
7. Using 10 ml pipette protoplasts were transported to 50 ml tubes and put on ice.
8. Protoplast were centrifuged for 3 min (150 x g, 4 °C) and washed twice with W5 buffer. Be careful not to resuspend protoplast to abruptly. After second wash step protoplast were resuspended in 1 ml of MMg solution. Protoplasts were calculated with help of hemocythometer and diluted to optimal concentration of 2 x 10⁴ cells in 100 μl with MMg solution (see Notes 5-7).
9. 5-10 μg plasmid DNA coding a tag-protein fusion was alequoted to 2 ml eppendorf tube (see Note 8).
10. 100 μl of isolated protoplasts in MMg medium were transferred to the tube using a pipette tip with the tip of the tip cut off.
11. Using a pipette tip with the tip of the tip cut off 110 μl of PEG solution was added. Solution was gently mixed and left for 15 min at RT in horizontal position (see Note 9).
12. After incubation, 450 μl of W1 buffer was added to the tubes to dilute PEG solution, mixed and centrifuged for 3 min, 300 x g.
13. Supernatant was removed and harvested protoplasts were resuspended in 300 μl of W1 solution.
14. Transfected protoplasts were incubated overnight at 22 °C in horizontal position in the dark for protein expression. It is important not to disturb protoplasts at this stage.
15. From this point protoplasts can be used in various protein analysis techniques (kinase assay, in vivo protein degradation assay, protein subcellular localization, BiFC and FRET analyses) (Ludwikow et al., 2014). Here we show in vivo degradation assay for plant proteins.
In vivo degradation assay in protoplasts
1. After overnight incubation (as indicated in step A14) in the dark protoplasts were treated with 50 μM MG132 (proteasome inhibitor) or mock treated with 0.1% DMSO for 6 h.
2. After brief centrifugation (300 x g, RT) supernatant was removed and cells were resuspended and disrupted in 100 μl of protein isolation buffer.
3. Protein concentration was determined using a NanoDrop spectrophotometer.
4. Prepared samples were separated by SDS-PAGE and further analyzed by Western blotting.

Representative data

Figure 2. MG132 treatment increases ACC synthase6 protein accumulation in the abi1td protoplasts

Protoplasts isolated from WT Col-0 and the ABI1 knockout line (abi1td) were transformed with 5 µg of DNA plasmid coding for StrepTag-ACS6. Transformed protoplasts were treated with 50 µM MG132 or an equivalent volume of DMSO (mock control) for 6 h prior harvesting. A Western blot with anti-StrepTag antibodies confirms the presence of the StrepTag-ACS6 protein. The Western blot shown is representative of at least three independent experiments. Coomassie staining confirms equal protein loading (Ludwikow et al., 2014).

Notes

This protocol is applicable to Brassica napus protoplast isolation and transformation.
To avoid mesophyll cells damage (visible as dark green spots) don't use too much pressure when applying the tape.
It is not recommended to digest leaves for more than 60 min. Protoplasts yield at this point will not increase, but they lose viability.
If one hour digestion did not freed all mesophyll cells from tape fragments, one can gentle dip tape a few times in enzyme solution, to increase protoplast yield.
Use swinging bucket rotor for centrifugation.
Keep low acceleration and deceleration values during centrifugation.
Resuspend the protoplasts by gently rocking the tube.
Use hemocytometer to achieve accurate and reproducible results.
A large volume of plasmid DNA decreases transformation efficiency, therefore keep the volume around 10 μl. Low transformation efficiency is usually a result of low quality plasmid DNA.

Recipes

Enzyme solution (10 ml)
1.2% cellulose
0.4% macerozyme
0.4 M mannitol
20 mM KCl
20 mM MES
dH₂O
Filter sterilize
Incubate at 55 °C for 10 min
Prepare fresh, do not store
W5 (50 ml)
154 mM NaCl
125 mM CaCl₂
5 mM KCl
2 mM MES
dH₂O
Filter sterilize, autoclave
Stored at 4 °C
W1 (10 ml)
0.5 M mannitol
20 mM KCl
4 mM MES
dH₂O
Filter sterilize, autoclave
Stored at 4 °C, but no longer than 2 weeks
MMg (10 ml)
0.4 M mannitol
15 mM MgCl₂
4 mM MES
dH₂O
Filter sterilize, autoclave
Stored at 4 °C, but no longer than 2 weeks
40% PEG (10 ml)
4 g PEG 4000
200 mM mannitol
100 mM CaCl₂
Filter sterilize, autoclave
Stored at 4 °C, but no longer than 3 weeks
Protein isolation buffer
20 mM HEPES (pH 7.5)
10 mM MgCl₂
1 mM DTT
1 mM PMSF
Protease inhibitor
dH₂O
Stored at -20 °C

Acknowledgments

This work was supported by COST Action FA0605 project 682/N-COST/2010/0, the National Science Centre grants (5615/B/P01/2010/39, DEC-2012/05/B/NZ3/00352, DEC-2011/03/N/NZ3/01796) and POLAPGEN grant no. WND-POIG.01.03.01-00-101/08. This protocol was adapted from Wu et al. (2009).

References

Ludwików, A., Ciesla, A., Kasprowicz-Maluśki, A., Mitula, F., Tajdel, M., Galganski, L., Ziolkowski, P. A., Kubiak, P., Malecka, A., Piechalak, A., Szabat, M., Gorska, A., Dabrowski, M., Ibragimow, I. and Sadowski, J. (2014). Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis. Mol Plant 7(6): 960-976.
Wu, F. H., Shen, S. C., Lee, L. Y., Lee, S. H., Chan, M. T. and Lin, C. S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5: 16.