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Plant Detectives Manual: a research-led approach for teaching plant science

Activity 6: Quantification of anthocyanins

6.1) Introduction and objectives

Anthocyanins are a subset of a large group of plant phenolics called flavonoids. These water-soluble pigments accumulate in the vacuole and play protective roles during acclimation to high light. Specifically, anthocyanins are glycosylated flavonoids and are responsible for the pink, purple and blue colours in plant organs (Fig. 5) (Lee et al. 2008). The colours are determined by the chemical groups attached to the ring B of the basic flavonol skeleton and the pH of the vacuole. Anthocyanins accumulate during stress conditions for example, such as a lack of nitrogen. Thus the presence or absence of anthocyanins can be used as an indicator for stress responses within the plant.

 

You will extract total pigment from frozen, ground leaf tissue from four-week-old wild type and mutant plants. You will then measure absorbance, quantify anthocyanins, and analyse the spectrum of the extract in the range of 400–800 nanometres (nm).

 

The main objectives of Activity 6 are to:

  1. isolate total anthocyanins from both wild type and mutant plants grown in the light using a simple organic solvent extraction procedure
  2. quantify anthocyanins using their spectrophotometric properties.

 

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6.2) Materials

  1. 1% (v/v) HCl
  2. 1.5 millilitre (ml) Eppendorf tubes
  3. 70% (v/v) methanol
  4. centrifuge (tabletop or microcentrifuge)
  5. chemical hood
  6. chloroform
  7. gloves
  8. goggles, one per person
  9. liquid nitrogen (–197 °C!!!)
  10. MilliQ water
  11. P1000 and P200 micropipettes, and tips (one each)
  12. pestle for grinding; alternatively, a mortar and pestle could be used
  13. plate reader BIO-TEK uQuant and 96 well plate or Spectrophotometer and cuvette
  14. vortex (one per class or one per group)

6.3) Procedure

Using the protocol below (based on published methods by (Lee et al. 2005) and (Neff and Chory 1998)) you will extract anthocyanins and quantify their content in your leaf tissues. Note that teaching staff will perform the harvesting. Depending on the type of grinding method, you will be asked to continue from Step 3 or Step 4.

6.3.1) Anthocyanin extraction

Materials

Method

Eppendorf tubes

  1. Harvest approximately 30 milligrams of leaf tissue from FOUR wild type and mutant plants and place each into a 1.5 ml Eppendorf tube. Make sure tubes are properly labelled beforehand. TIP: label tubes with your group number, genotype, and replicate. For example, 01–WT–02 is the replicate number two of the wild type leaf tissue of Group 1.

Goggles, gloves, liquid nitrogen

  1. Freeze the material immediately by dipping the sample in liquid nitrogen. IMPORTANT: wear goggles and gloves to avoid burning your skin. Liquid nitrogen boils at −196 °C (very cold!) and causes rapid freezing when in contact with living tissue. Your eyes are very vulnerable to liquid nitrogen. EXERCISE CARE and consider your surroundings and anyone nearby.

Pestle for grinding

  1. Grind the frozen tissue.

Pipettes and tips, methanol, vortex

  1. Add 400 microlitres (µl) of acidified methanol (70% methanol; 1% (v/v) HCl) to your ground samples.

Vortex

  1. Vortex the samples vigorously for 30 seconds.

Fume hood, chloroform, water, vortex

  1. Add 700 µl of chloroform and 300 µl of H2O to the sample.

Vortex

  1. Vortex the samples for ten seconds.

Centrifuge

  1. Centrifuge at full speed (13,000 revolutions per minute) for five minutes.4

6.3.2) Anthocyanin quantification

Materials

Method

Eppendorf tubes

  1. Transfer as much as possible (~500 µl) of the upper (aqueous) phase to a new tube. If you are not sure about the recovered volume, you can estimate it using a pipette. Ask your demonstrator.

Plate reader or spectrophotometer, plate or cuvette.

  1. Use 200 µl of the ‘blank’ solution5 supplied by the plate reader operator along with your samples to read absorbance using the plate reader.

 

  1. Measure sample absorbance at 530 and 657 nm. The operator will enter the individual absorbance readings into an Excel spreadsheet.

 

  1. Calculate the anthocyanin concentration on a tissue gram basis as:

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Where:

ε = the molar absorptivity constant of cyanidine–3–glucoside (26900 L cm–1 mol–1); monomeric anthocyanins can be expressed as cyanidine–3–glucoside.

l = path length: one centimetre (cm); however this may need to be recalculated when using the plate reader.

MW: molecular weight of the cyanidine–3–glucoside is 449.2 g mol−1

DF: Assuming that 500 µl were extracted in Step 9, and that most of the anthocyanin fractionated in the methanol–water fraction (400 µl + 300 µl), DF equals 700/500=1.4.

Volume = volume of the aqueous phase extracted on Step 4 in ml (0.4 ml)

FW = tissue mass in grams (g)

 

  1. If the colour of your extract is reddish, then take 200 µl to a new tube and add 10 µl of 1N NaOH. Observe what happens with the colour. If nothing happens add more NaOH. You can do this for one or both samples.

6.4) Expected outcomes

  1. Calculate the amount of anthocyanins in both genotypes in micrograms (µg) of pigment per gram (g) of tissue (µg g–1).
  2. Also, note whether there are any obvious differences in ‘reddish’ colour in the rosette leaves, or under the rosette, between different genotypes?
  3. Is there a noticeable stress trait that can be correlated with the anthocyanin content (i.e., a leaf that is more wilted has a different anthocyanin content to one that is not)?

 


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