Transient Expression Assay Using Arabidopsis Mesophyll Proto

Thursday, December 04, 2003

Description
Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts :
PEG Transfection

Procedure
All steps are carried out at 23oC
Add 20 ml DNA (20-40 mg of plasmid DNA of 5 kb in size)
Add 200 ml protopolasts to a microfuge tube (4 x 104 protoplasts), mix well
Add 220 ml of PEG/Ca solution, mix well (handle 6 samples each time)
Incubate at 23oC for 5-30 min
Dilute with 0.8 ml W5 solution, mix well
Spin at speed 3 in a Clinical centrifuge for 1 min, remove PEG
Resuspend protoplasts gently, dilute in 100 ml WI or W5, add to 1 ml WI or W5 in a 6-well plate
This procedure can be scaled up or down depending on the experimental needs

Due to the transient nature of the experiments, it is not necessary to perform experiments under sterile conditions. The addition of Amp (50 mg/ml) can prevent bacterial growth if it is necessary.
The system is most suitable for the study of early events in diverse signal transduction pathways, gene regulation, and cell death (Asai et al., Plant Cell 12:1823-1835, 2000).
The use of carrier DNA is unnecessary.
Use 6-well tissue culture dishes (Falcon 3046) for protoplast incubation. We now also use 12-well or 24-well plates for large experiments (with 50-200 samples).
The dish can be coated with 5% calf serum for 1 sec before use to prevent sticking of protoplasts to the plastic.
The protoplast incubation time is 2-6 h for RNA analysis and 2-12 h for enzyme activity analysis and protein labeling. About 100-103 protoplasts are sufficient for reporter enzyme assays, 104-105 protoplasts are required for protein labeling & immunoprecipitation or western blot analysis, and 106 protoplasts are necessary for RNA analysis.
These protoplasts can be cultured for cell wall regeneration and cell cycle initiation with proper medium and plant hormones (Damm et al., 1989, MGG 217: 6-12.; Masson & Paszkowski, 1992, Plant J. 2: 829-833).
PEG transformation efficiency is 50-90% based on GFP expression. If your protoplasts are healthy (from optimal leaf materials. Please test), most protoplasts remain intact.
Electroporation efficiency is 10-30% (depending on plant conditions). More than 50% protoplasts can be killed by electroporation. However conditions could be adjusted to reduce killing (Please test). Protoplasts produced from different plant species and tissues or growth conditions may react differently to electroporation. For instance, etiolated or greening maize mesophyll protoplasts tolerate electroporation extremely well (Sheen, Plant Cell, 3:225-245, 1991).

Harvest protoplasts by centrifugation at 100 x g for 2 min. Remove the supernatant. Freeze and store samples at -80oC until ready for analysis.
Add 100 ml of hypotonic buffer (10 mM Tris, pH 8 and 2 mM MgCl2) or LUC lysis buffer (Promega), vortex vigorously for 2 sec to lyse the protoplasts. (Note that the LUC lysis buffer contains 1% Triton X-100).
A fast and economical xylenes extraction protocol is used for CAT assay (Seed and Sheen, 1988, Gene 67, 271-277; Sheen, in the supplement 9.6.5 of the Current Protocols in Molecular Biology, Ausubel eds). Heating the cell extract at 65oC for 10 min in the presence of 5 mM EDTA might eliminate potential inhibitors for CAT activity (It seems to be useful for Col protoplasts but not C24). We use a Promega kit for LUC assay with a luminometer. The GUS assay has been described by Jefferson (add the cell extract into 100 ul of 1 mM MUG, incubate for 30-90 min at 37oC, add 0.9 ml 0.2 M Na2CO3 to stop the reaction, and measure the fluorescence of MU).

Recipes
Enzyme solution
1-1.5 % cellulase R10 (RS is too strong)
0.2-0.4% macerozyme R10 (Yakult Honsha, Tokyo, Japan)
0.4 M mannitol
20 mM KCl
20 mM MES, pH 5.7
Heat the enzyme solution at 55oC for 10 min (to inactivate proteases and enhance enzyme solubility) and cool it to room temperature before adding
10 mM CaCl2
5 mM b-mercaptoethanol (optional)
0.1% BSA (Sigma A-6793)
The enzyme solution is light brown but clear (passed through a 0.45 mm filter).

PEG solution (40%, v/v)
4 g PEG4000 (Fluka, #81240)
3 ml H2O
2.5 ml 0.8 M mannitol
1 ml 1M Ca(NO3)2 or CaCl2

Washing and incubation solution (WI)
0.5 M mannitol,
4 mM MES, pH 5.7
20 mM KCl

W5 solution
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES (pH 5.7) (no glucose since we use glucose as a signal)

MMg solution
0.4 M mannitol
15 mM MgCl2
4 mM MES (pH 5.7)

Electroporation

40 mg plasmid DNA
4-6 x 104 protoplasts/300 ml of 0.3 M mannitol/4 mM MES, pH 5.7/150 mM KCl
300-400 V/cm
5 msec, 200 mF, 1-2 pulses

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Transient Expression Assay Using Arabidopsis Mesophyll Proto