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Quantification of Anthocyanin Content   

Edited by
Ru Zhang
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In this protocol

Original research article

A brief version of this protocol appeared in:
The Plant Cell
May 2013

Abstract

Anthocyanins are a class of flavonoids and important plant pigments. They attract insects to pollinate flowers, protect plants from UV irradiation, and act as antimicrobial agents against herbivores and pathogens. Biosynthesis of anthocyanin is stimulated by diverse developmental signals and environmental stresses including drought, wounding, pathogen infection and insect attack. Plant hormones such as jasmonates, a stress-related plant hormone, also induce accumulation of anthocyanins. Sensitivity of plants to these stress stimuli can be measured by accumulation of anthocyanins. Here we describe a simple method for measurement of anthocyanins in Arabidopsis thaliana seedlings. Amount of anthocyanins are calculated only from absorbances at 530 and 657 nm of crude extract.

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.
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Nakata, M. and Ohme-Takagi, M. (2014). Quantification of Anthocyanin Content. Bio-protocol 4(7): e1098. DOI: 10.21769/BioProtoc.1098.
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mohamad padri
Naresuan University
dear authors,

I would like to ask about what kind of Volume do we use that you mention in the 7th step of procedure ?
secondly, based on the calculation, the result will be in Abs530/g F.W. would you mind to exemplify this ?

many thanks

best regards
2/15/2016 10:42:38 PM Reply