A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips

Materials and Reagents

1. Square dishes for tissue culture [External dimension (mm): 126.40 x 126.40 x 20.00] (SPL Life Sciences, catalog number: 10125)
2. 1.5 ml micro-tubes (Eppendorf, catalog number: 0030121589)
3. Microscope slides (dimensions: 76 x 26 mm; thickness: 1 mm) (Marienfeld, catalog number: 1000200)
4. Transparent slides coverslips (dimensions: 60 x 24 mm; thickness: 0.170 mm ± 0.005 mm) (Marienfeld, catalog number: 0107242)
5. Arabidopsis thaliana
6. Diphenylboric acid-2-aminoethyl ester (DPBA) (Sigma-Aldrich, catalog number: D9754 )
7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
8. Murashige & Skoog medium including B5 vitamins (Duchefa Biochemie, catalog number: M0231 )
9. Phyto Agar (Duchefa Biochemie, catalog number: P1003 )
10. Sucrose (Duchefa Biochemie, catalog number: S0809 )
11. DPBA staining solution (see Recipes)
    

Equipment

1. Tweezers
2. A plant growth chamber
3. Micro-tubes rotator
4. Pipette
5. Confocal microscopy system (Leica, model: SP8 )
   

Procedure

1. Plant growth and DPBA staining procedure
   1. Here, Arabidopsis thaliana is used. However, this protocol can be applied for other plants as well. Grow plants on MS medium supplemented with 2% sucrose and 1.2% phyto-agar for 5 days (Figure 1). The growth chamber conditions were 22 ± 1 °C, long-day (16 h light/8 h dark), and light intensity 100 μmol m⁻² s⁻¹.
      Note: After seeding, remember to place the medium plates vertically in the growth chamber. Thereby, the roots grow on the surface of the medium and intact root tip samples can be obtained.
       
      
      Figure 1. Five-day-old Arabidopsis seedlings. Scale bar = 5 mm.
      
      
   2. Add 1 ml of DPBA staining solution (Recipe 1) to each micro-tube.
   3. Use a clean tweezers to carefully transfer the 5-day-old Arabidopsis seedlings to the micro-tube containing DPBA staining solution (5 seedlings/micro-tube).
   4. Place the micro-tube in the rotator and rotate for 5 min at room temperature.
   5. Stop the rotator, transfer the micro-tube to a rack and remove the DPBA staining solution.
      Note: At this step, carefully use a pipette to remove the DPBA staining solution and try to not damage the plants, especially the root tips.
   6. For washing, add 1.5 ml of distilled water to each micro-tube. Place the micro-tube in the rotator and rotate for 2 min at room temperature.
   7. Stop the rotator, transfer the micro-tube to a rack and remove water.
   8. Repeat Steps A6 and A7 two more times.
      Note: After washing, can keep the plants in water and try to detect immediately. Do not leave the samples staying in water for longer than 30 min.
   9. Transfer the seedlings to a microscope slide, cover it and detect the flavonols accumulation in the root tips by a confocal microscope.
      
      
      
2. Confocal microscopy
   1. For DPBA-kaempferol, apply the emission spectrum (475-500 nm).
   2. For DPBA-quercetin, apply the emission spectrum (585-619 nm).
   3. Remember to take an additional bright field picture for control.

Data analysis

Arrange the data as following order: (1) Bright field; (2) Kaempferol; and (3) Quercetin. Please check some previous publications for details (Nguyen et al., 2013, 2015 and 2016; Vu et al., 2015).


Figure 2. Accumulation of flavonols (kaempferol and quercetin) in the root tips of 5-day-old Arabidopsis seedlings (wild-type). Scale bar = 100 μm.

Recipes

1. DPBA staining solution
   

   Reagent	Amount for 100 ml
   DPBA	0.25 g
   Triton X-100	20 μl
   Milli-Q water	up to 100 ml

   Notes:
   1. Some crystals of DPBA can be retained and seen after mixing.
   2. This staining solution can be stored at -20 °C for further uses.

Acknowledgments

I would like to appreciate Dr. Cuong Thach Nguyen (Nguyen Tat Thanh University, Vietnam) and Dr. Minh Tan Nguyen (UCLA School of Dentistry, US) for their critical reading this protocol. This protocol was derived from previous publications (Nguyen et al., 2013, 2015 and 2016). The authors declare that they have no conflict of interest.

References

1. Lewis, D. R., Ramirez, M. V., Miller, N. D., Vallabhaneni, P., Ray, W. K., Helm, R. F., Winkel, B. S. and Muday, G. K. (2011). Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks. Plant Physiol 156(1): 144-164.
2. Nguyen, H. N., Kim, J. H., Hyun, W. Y., Nguyen, N. T., Hong, S. W. and Lee, H. (2013). TTG1-mediated flavonols biosynthesis alleviates root growth inhibition in response to ABA. Plant Cell Rep 32(4): 503-514. 
3. Nguyen, N. H., Jeong, C. Y., Kang, G. H., Yoo, S. D., Hong, S. W. and Lee, H. (2015). MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J 84(6): 1192-1205. 
4. Nguyen, N. H., Kim, J. H., Kwon, J., Jeong, C. Y., Lee, W., Lee, D., Hong, S. W. and Lee, H. (2016). Characterization of Arabidopsis thaliana FLAVONOL SYNTHASE 1 (FLS1) -overexpression plants in response to abiotic stress. Plant Physiol Biochem 103: 133-142. 
5. Sheahan, J. J. and Rechnitz, G. A. (1992). Flavonoid-specific staining of Arabidopsis thaliana. Biotechniques 13(6): 880-883.
6. Vu, T. T., Jeong, C. Y., Nguyen, H. N., Lee, D., Lee, S. A., Kim, J. H., Hong, S. W. and Lee, H. (2015). Characterization of Brassica napus Flavonol Synthase Involved in Flavonol Biosynthesis in Brassica napus L. J Agric Food Chem 63(35): 7819-7829.