Materials and Reagents

Pipette tips
1.5 ml Eppendorf tubes
Sterilized filter paper
Plastic Petri dishes for plant culture
Slide
Cover glass
0.45 micron filter
Aluminum foil
Arabidopsis thaliana seeds of ROP7pro:GUS and RopGEF4pro:GUS lines
100%, 75%, 40%, 20%, 10%, 5% ethanol in water
50% glycerol (Sangon Biotech, catalog number: A100854 )
100% methanol
37% hydrochloric acid (12 N)
Sodium hydroxide (AMRESCO, catalog number: 0583 )
Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0322 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Potassium ferricyanide (AMRESCO, catalog number: 0713 )
Potassium ferrocyanide (Sigma-Aldrich, catalog number: P9387 )
X-Gluc (Sigma-Aldrich, catalog number: B5285 )
Dimethylformamide (AMRESCO, catalog number: 0464 )
Sodium dihydrogen phosphate (NaH₂PO₄·H₂O) (Sigma-Aldrich, catalog number: S9638 )
Disodium hydrogen phosphate (Na₂HPO₄·7H₂O) (Sigma-Aldrich, catalog number: 431478 )
20% methanol in 0.24 N hydrochloric acid (see Recipes)
60% ethanol in 7% sodium hydroxide (see Recipes)
GUS staining solution (see Recipes)

Equipment

Pipetman 100 μl (Gilson, catalog number: F123615 )
Pipetman 1,000 μl (Gilson, catalog number: F123602 )
Tweezers
Brush pen
Plant growth chamber (Percival Scientific, model: CU-36L5 ) and greenhouse
Pots
Incubator at 37 °C (SANFA, model: DNP-9052 )
Microscope (ZEISS, model: Axio Imager A1 )
Water purification system (deionized water) (EMD Millipore, model: Elix^® Essential , 5 L)

Procedure

Arabidopsis plants were grown according to Arabidopsis growing guide http://www.bio-protocol.org/e126.
Harvest the fully expanded rosette leaves from 3-4 week-old plants, and peel the epidermal strips from the abaxial surface of the leaf (Figure 1A), remove the mesophyll tissue from the strips with a brush pen (Figure 1B). Epidermal strips or leaf are then immersed in 1.5 ml Eppendorf tubes with GUS staining solution (see Recipes), and keep the tubes in an incubator at 37 °C under darkness for 16-20 h.

Figure 1. Images show the peeling of the epidermis from the abaxial surface of leaf (A) and removing the mesophyll tissue from epidermal strip by a brush pen (B)
Replace the supernatant with 1 ml 100% ethanol and incubate for 30 min, repeat this step for 2 times.
Replace the supernatant with 1 ml 75% ethanol, and incubate for 5 min.
Replace the supernatant with 1 ml 20% methanol in 0.24 N hydrochloric acid, and incubate at 37 °C for 15 min.
Replace the supernatant with 1 ml 60% ethanol in 7% sodium hydroxide, and incubate for 15 min.
Replace the supernatant with 1 ml 40% ethanol, and incubate for 5 min; replace the supernatant with 20%, 10% and 5% ethanol gradually each for 5 min. After all these steps, the leaf tissue will turn to white, and keep the samples in 5% ethanol.
Put one drop of 50% glycerol on the slide, and place the samples in the glycerol solution; put the cover glass on the samples.
Observe the samples under a microscope and take pictures:
1. Switch on the light source of the microscope, and rotate the nosepiece to the lowest-power objective.
2. Place the slide on the stage of the microscope, and move the slide to center the specimen under the lens.
3. Adjust the large coarse focus knob until the specimen is in focus.
4. Scan the slide at low power objective (4x or 10x objective), center the part of the specimen that one wants to view, and then rotate the nosepiece to the 20x or 40x objective.
5. Adjust the small fine focus knob until the cells are in focus, and adjust the diaphragm until the cells have clear and sharp contrast.
6. Take a picture with the CCD.

Data analysis

As shown in Figure 2, the GUS signals in guard cells were very clear in the images of GUS staining from both the leaf (Figure 2C) and the epidermal strips (Figure 2D) of RopGEF4pro:GUS lines. The results were consistent with the specific expression of RopGEF4 in guard cells reported by Li and Liu (2012). However, the GUS signal was not clear when stained the whole leaf of ROP7pro:GUS line because of the strong background of mesophyll tissue (Figure 2A). When the epidermal strips were separated from mesophyll cells, we can see a clear guard cell GUS signal after GUS staining (Figure 2B). When this method is applied, it is better to use 3-4 week old plants of Arabidopsis, because the epidermal strips are easily peeled from the mesophyll tissue during this time. This method is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.

Figure 2. GUS staining with the leaf (A) and epidermal strips (B) from ROP7pro:GUS line, and leaf (C) and epidermal strips (D) from RopGEF4pro:GUS line. Scale bar = 10 μm.

Notes

Please be very careful to keep the epidermal strips in the tubes in each step, try to avoid losing the epidermal strips when removing the supernatant.
The epidermal strips became soft and easily broken after GUS staining procedure, so be very careful to flatten the epidermal strip on the slide.

Recipes

20% methanol in 0.24 N hydrochloric acid
Mix:
20 ml of 100% methanol
2 ml of 37% hydrochloric acid (12 N)
Deionized water 78 ml
60% ethanol in 7% sodium hydroxide
Dissolve 28 g sodium hydroxide in deionized water to 100 ml to make 28% sodium hydroxide
Mix:
60 ml of 100% ethanol
25 ml of 28% sodium hydroxide
15 ml of deionized water
GUS staining solution
100 mM sodium phosphate buffer (pH = 7.0)
10 mM EDTA
0.1% Triton X-100
0.5 mM potassium ferricyanide
0.5 mM potassium ferrocyanide
1 mM X-Gluc
1. Dissolve 0.8892 g X-Gluc in 100 ml dimethylformamide to make a 20 mM stock and keep in the dark at -20 °C
2. Make 200 mM sodium phosphate buffer (pH 7.0): dissolve 1.08 g sodium dihydrogen phosphate (NaH₂PO₄·H₂O) and 3.27 g disodium hydrogen phosphate (Na₂HPO₄·7H₂O) in deionized water up to 100 ml
3. Mix:
  50 ml of 200 mM sodium phosphate buffer (pH 7.0)
  2 ml of 500 mM EDTA solution
  0.5 ml of 20% (w/v) Triton X-100
  1 ml of 50 mM potassium ferricyanide
  1 ml of 50 mM potassium ferrocyanide
  5 ml of 20 mM X-Gluc
4. Bring up to 100 ml with deionized water
5. Filter-sterilize (0.45 micron filter) and keep at 4 °C wrapped with aluminum foil

Acknowledgments

This work was supported by the National Science Foundation of China (Grant No. 31571450 to Y.-L.C.). The protocol was modified according to Pecinka et al. (2009).

References

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Li, Z. and Liu, D. (2012). ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis. FEBS Lett 586(9): 1253-1258.
Li, Z., Kang, J., Sui, N. and Liu, D. (2012). ROP11 GTPase is a negative regulator of multiple ABA responses in Arabidopsis. J Integr Plant Biol 54(3): 169-179.
Mustilli, A. C., Merlot, S., Vavasseur, A., Fenzi, F. and Giraudat, J. (2002). Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14(12): 3089-3099.
Pecinka, A., Rosa, M., Schikora, A., Berlinger, M., Hirt, H., Luschnig, C. and Mittelsten Scheid, O. (2009). Transgenerational stress memory is not a general response in Arabidopsis. PLoS One 4(4): e5202.
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Takemiya, A., Sugiyama, N., Fujimoto, H., Tsutsumi, T., Yamauchi, S., Hiyama, A., Tada, Y., Christie, J. M. and Shimazaki, K. (2013). Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening. Nat Commun 4: 2094.
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