Quantification of Anthocyanin Content

Materials and Reagents

1. Arabidopsis thaliana seedlings (~10 days after germination)
   Note: Amount of anthocyanin per seedling weight is higher in young seedlings.
   
2. Bleach solution
   
3. Sterile dH₂O
   
4. Methanol
   
5. Acetic acid
   
6. Murashige and Skoog medium salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
   
7. Sucrose
   
8. 2-Morpholinoethanesulfonic acid (MES)
   
9. Agar (for plant culture)
   
10. Modified Murashige and Skoog medium (see Recipes)
    
11. Extraction buffer (see Recipes)
    

Equipment

1. Paper towel
   
2. Spectrophotometer
   
3. Microcentrifuge
   
4. Microcentrifuge tubes
   
5. Mortar and pestle
   
6. Liquid nitrogen
   
7. Electric balance
   

Procedure

1. Add bleach solution to Arabidopsis thaliana seeds and shake gently for 7 min.
   
2. Rinse three to five times with sterile dH₂O.
   
3. Sow surface-sterilized seeds on modified MS medium.
   
4. Grow the plants under long day (16 h light/8 h dark) or continuous light condition (50~70 µmol/m²/s) at 22 °C for 7~10 days.
   
5. Measure fresh weight of 10~15 seedlings.
   Note: Remove surface moisture with paper towel before measurement.
   
6. Freeze seedlings with liquid nitrogen and grind in a mortar and pestle.
   
7. Add 5 volumes (based on fresh weight) of extraction buffer and mix thoroughly.
   
8. Centrifuge at 12,000 x g for 5 min at room temperature.
   
9. Transfer supernatant to new tube.
   
10. Centrifuge at 12,000 x g for 5 min at room temperature.
    
11. Transfer supernatant to new tube.
    
12. Measure absorbances at 530 and 637 nm.
    
13. Calculate anthocyanin content (Abs₅₃₀/g F.W.) by [Abs₅₃₀ - (0.25 x Abs₆₅₇)] x 5.
    Note: To correct contribution of chlorophyll to the absorbances at 530 nm, the formula Abs530 - (0.25 x Abs657) was used. Multiplication of “5” can be changed depending on volumes of added extraction buffer in step 5. For example, if 10 volumes of extraction buffer was added, multiply “10”.
    

Recipes

1. Modified Murashige and Skoog medium
   1x Murashige and Skoog medium salt
   0.5% Sucrose
   0.05% MES
   Adjust pH to 5.7 with KOH
   0.8% agar
   
2. Extraction buffer
   45% methanol
   5% acetic acid
   

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References

1. Nakata, M., Mitsuda, N., Herde, M., Koo, A. J., Moreno, J. E., Suzuki, K., Howe, G. A. and Ohme-Takagi, M. (2013). A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell 25(5): 1641-1656.