Quantification of Callose Deposition in Plant Leaves    

Materials and Reagents

1. Plant materials: Arabidopsis thaliana cotyledons and leaves, Solanum lycopersicum leaves or Citrus leaves
   
2. 96% ethanol
   
3. Na₂HPO₄^.2H₂O
   
4. NaH₂PO₄^.2H₂O
   
5. Methyl blue (Sigma-Aldrich, catalog number: M6900 )
   
6. Sodium phosphate buffer (see Recipes)
   
7. Methyl blue solution 0.5% and 0.05% (see Recipes)
   
   

Optional

1. Trizma^® base (Sigma-Aldrich, catalog number: T1503 )
   
2. Fluorescent Brightener 28 (synonym Calcofluor white) (Sigma-Aldrich, catalog number: F3543 )
   
3. HCl
   
4. Tris^.HCl buffer (see Recipes)
   
5. Fluorescent brightener solution 0.01% (see Recipes)
   

Equipment

1. Leica IRB epifluorescence microscope with UV filter (BP 340 to 380 nm, LP 425 nm) equipped with a Leica DC300F camera (Leica Microsystems, model: Leica IRB )

Software

1. Digital photographs analysis software like GIMP (http://www.gimp.org) or Adobe Photoshop (adobe.com) are recommended

Procedure

Note: Staining with fluorescent Brightener is optional. This will stain the chitin present in the sample, and can be useful to stain the fungus. However, the use of this staining can mask the fluorescence emitted by callose. For this reason, if the goal of the experiment is to quantify callose deposition, the use of fluorescent Brightener is not recommended.

1. Staining procedure
   
   1. This procedure can be used for staining Arabidopsis thaliana cotyledons and leaves, Solanum lycopersicum leaves or Citrus leaves with little variation according to tissue toughness.
      
   2. Sample the entire leaves or the cotyledons from the plant and place them in 50 ml tubes containing 96% ethanol for chlorophyll removal. Distain the samples in 96% ethanol until they appear completely white (time can vary between 1 day for small leaves like the ones of Arabidopsis till one week for citrus leaves). The saturated distaining 96% ethanol can be replaced, if necessary.
      
   3. Rehydrate the samples in sodium phosphate buffer for 30 min.
      
   4. Discard the phosphate buffer and cover the sample with 0.05% Methyl blue solution.
      Optional: Add 1/5 of fluorescent brightener solution (1 ml per each 4 ml of methyl blue solution used in the previous step).
      
   5. Incubate for 30 min and discard the solution.
      
   6. For Arabidopsis cotyledons go to step A8. Callose can be observed after 3-4 h of incubation with 0.05% Methyl blue as previously described by Luna et al. (2011).
      
   7. Cover the sample with 0.5% Methyl blue solution and incubate for 24 h in darkness. (Time may vary depending on the sample. Thought samples, such as citrus leaves, may require a vacuum stroke of 2 min and up to 7 days of staining.)
      
   8. For microscope observation, mount the samples on slides with the adaxial surface up using freshly prepared 0.05% Methyl blue.
      
   9. Observe the samples with an epifluorescence microscope with UV filter. Stained callose appears as fluorescent yellow spots (Figure 1).
      Note: Be aware to use all the time the same augmentation.
      
      
      Figure 1. Representative picture of callose deposition in tomato leaves upon Botrytis cinerea infection with fluorescent brightener solution (A) and without fluorescent brightener solution (B). The arrow highlights callose. Representative picture of control leaves (C).
   
   
2. Image processing
   
   1. Open the images with the photography software (Figure 2).
      
      
      Figure 2. Yellow spots corresponding to stained callose analyzed for the number of pixels using A. GIMP (GNU Image Manipulation Program) or B. Adobe Photoshop
      
      
   2. Select the brilliant yellow pixels that correspond to callose using a Magic wand tool or select by color range.
      
   3. Use the histogram window to determine the number of pixels.
      
   4. Represent callose intensity as average number of bright pixels/number of total pixels.
      In the case of cotyledons it is possible to calculate the yellow pixels per leaf area.
      See Video for more details.
      
      
      
      Video 1. Quantification process using Gimp
      
      
      
      Video 2. Quantification process with photoshop

Recipes

1. Sodium phosphate buffer (0.07 M, pH=9)
   Dissolve 12.46 g of Na₂HPO₄^.2H₂O in 1 L of distilled water
   Dissolve 0.966 g of NaH₂PO₄^.2H₂O in 100 ml of distilled water
   Adjust the pH of Na₂HPO₄^.2H₂O solution to 9, using the solution of NaH₂PO₄^.2H₂O
   
2. Methyl blue solution freshly prepared
   For 0.5% solution: Dissolve 0.5 g of Methyl Blue in 100 ml of sodium phosphate buffer
   For 0.05% solution: Dissolve 0.05 g of Methyl Blue in 100 ml of sodium phosphate buffer
   
   

Optional solutions

3. Tris.HCl buffer (0.1 M, pH =8.5)
   Dissolve 0.6057 g of Tris base in 50 ml of distilled water and adjust the pH at 8.5 with HCl
   
4. Fluorescent brightener solution
   Dissolve 0.0125 g of Fluorescent Brightener 28 in 50 ml of Tris buffer
   Stored the solution in dark at room temperature
   

Acknowledgments

Authors thank Universitat Jaume I and the National R&D Plan (AGL2010-22300-C03-02, Spain for funding support.

References

1. Chen, X. Y. and Kim, J. Y. (2009). Callose synthesis in higher plants. Plant Signal Behav 4(6): 489-492.
   
2. Luna, E., Pastor, V., Robert, J., Flors, V., Mauch-Mani, B. and Ton, J. (2011). Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 24(2): 183-193.